Treatment of lipid transport and metabolism gene related diseases by inhibition of natural antisense transcript to a lipid transport and metabolism gene

ABSTRACT

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of a Lipid transport and metabolism gene, in particular, by targeting natural antisense polynucleotides of a Lipid transport and metabolism gene. The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of a Lipid transport and metabolism genes.

The present Application is a Divisional of U.S. Ser. No. 13/318,713filed Nov. 3, 2011, which is a 371 of International Application No.PCT/US2010/033908 filed May 6, 2010, which claims the priority to U.S.Provisional Patent Application Nos. 61/175,930 filed May 6, 2009,61/176,267 filed May 7, 2009, 61/180,646 filed May 22, 2009, 61/235,227filed Aug. 19, 2009, and 61/248,212 filed Oct. 2, 2009, which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

Embodiments of the invention comprise oligonucleotides modulatingexpression and/or function of a Lipid transport and metabolism gene andassociated molecules.

BACKGROUND

DNA-RNA and RNA-RNA hybridization are important to many aspects ofnucleic acid function including DNA replication, transcription, andtranslation. Hybridization is also central to a variety of technologiesthat either detect a particular nucleic acid or alter its expression.Antisense nucleotides, for example, disrupt gene expression byhybridizing to target RNA, thereby interfering with RNA splicing,transcription, translation, and replication. Antisense DNA has the addedfeature that DNA-RNA hybrids serve as a substrate for digestion byribonuclease H, an activity that is present in most cell types.Antisense molecules can be delivered into cells, as is the case foroligodeoxynucleotides (ODNs), or they can be expressed from endogenousgenes as RNA molecules. The FDA recently approved an antisense drug,VITRAVENE™ (for treatment of cytomegalovirus retinitis), reflecting thatantisense has therapeutic utility.

SUMMARY

This Summary is provided to present a summary of the invention tobriefly indicate the nature and substance of the invention. It issubmitted with the understanding that it will not be used to interpretor limit the scope or meaning of the claims.

In one embodiment, the invention provides methods for inhibiting theaction of a natural antisense transcript by using antisenseoligonucleotide(s) targeted to any region of the natural antisensetranscript resulting in up-regulation of the corresponding sense gene.It is also contemplated herein that inhibition of the natural antisensetranscript can be achieved by siRNA, ribozymes and small molecules,which are considered to be within the scope of the present invention.

One embodiment provides a method of modulating function and/orexpression of a Lipid transport and metabolism gene polynucleotide inpatient cells or tissues in vivo or in vitro comprising contacting saidcells or tissues with an antisense oligonucleotide 5 to 30 nucleotidesin length wherein said oligonucleotide has at least 50% sequenceidentity to a reverse complement of a polynucleotide comprising 5 to 30consecutive nucleotides within nucleotides 1 to 1299 of SEQ ID NO: 8, 1to 918 of SEQ ID NO: 9, 1 to 1550 of SEQ ID NO: 10, 1 to 329 of SEQ IDNO: 11, 1 to 1826 of SEQ ID NO: 12, 1 to 536 of SEQ ID NO: 13, 1 to 551of SEQ ID NO: 14, 1 to 672 of SEQ ID NO: 15, 1 to 616 of SEQ ID NO: 16,1 to 471 of SEQ ID NO: 17, 1 to 707 of SEQ ID NO: 18, 1 to 741 of SEQ IDNO: 19, 1 to 346 of SEQ ID NO: 20, 1 to 867 of SEQ ID NO: 21, 1 to 563of SEQ ID NO: 22 (FIG. 3) thereby modulating function and/or expressionof the Lipid transport and metabolism gene polynucleotide in patientcells or tissues in vivo or in vitro.

In another preferred embodiment, an oligonucleotide targets a naturalantisense sequence of a Lipid transport and metabolism genepolynucleotide, for example, nucleotides set forth in SEQ ID NO: 8 to22, and any variants, alleles, homologs, mutants, derivatives, fragmentsand complementary sequences thereto. Examples of antisenseoligonucleotides are set forth as SEQ ID NOS: 23 to 263 (FIGS. 4 to 9).

Another embodiment provides a method of modulating function and/orexpression of a Lipid transport and metabolism gene polynucleotide inpatient cells or tissues in vivo or in vitro comprising contacting saidcells or tissues with an antisense oligonucleotide 5 to 30 nucleotidesin length wherein said oligonucleotide has at least 50% sequenceidentity to a reverse complement of the an antisense of the Lipidtransport and metabolism gene polynucleotide; thereby modulatingfunction and/or expression of the Lipid transport and metabolism genepolynucleotide in patient cells or tissues in vivo or in vitro.

Another embodiment provides a method of modulating (unction and/orexpression of a Lipid transport and metabolism gene polynucleotide inpatient cells or tissues in vivo or in vitro comprising contacting saidcells or tissues with an antisense oligonucleotide 5 to 30 nucleotidesin length wherein said oligonucleotide has at least 50% sequenceidentity to an antisense oligonucleotide to a Lipid transport andmetabolism gene antisense polynucleotide; thereby modulating functionand/or expression of the Lipid transport and metabolism genepolynucleotide in patient cells or (issues in vivo or in vitro.

In a preferred embodiment, a composition comprises one or more antisenseoligonucleotides which bind to sense and/or antisense Lipid transportand metabolism gene polynucleotides.

In another preferred embodiment, the oligonucleotides comprise one ormore modified or substituted nucleotides.

In another preferred embodiment, the oligonucleotides comprise one ormore modified bonds.

In yet another embodiment, the modified nucleotides comprise modifiedbases comprising phosphorothioate, methylphosphonate, peptide nucleicacids, 2′-O-methyl, fluoro- or carbon, methylene or other locked nucleicacid (LNA) molecules. Preferably, the modified nucleotides are lockednucleic acid molecules, including .alpha.-L-LNA.

In another preferred embodiment, the oligonucleotides are administeredto a patient subcutaneously, intramuscularly, intravenously orintraperitoneally.

In another preferred embodiment, the oligonucleotides are administeredin a pharmaceutical composition. A treatment regimen comprisesadministering the antisense compounds at least once to patient; however,this treatment can be modified to include multiple doses over a periodof time. The treatment can be combined with one or more other types oftherapies.

In another preferred embodiment, the oligonucleotides are encapsulatedin a liposome or attached to a carrier molecule (e.g. cholesterol, TATpeptide).

Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of real time PCR results showing the foldchange+standard deviation in ABCA1 mRNA after treatment of 518A2 cellswith siRNA oligonucleotides introduced using Lipofectamine 2000, ascompared to control. Real time PCR results show that the levels of ABCA1mRNA in 518A2 cells are significantly increased 48 h after treatmentwith one of the siRNAs designed to ABCA1 antisense AK311445. Barsdenoted as CUR-0521, CUR-0519 and CUR-0523 correspond to samples treatedwith SEQ ID NOS: 23 to 25 respectively.

FIG. 1B is a graph of real lime PCR results showing the foldchanger-standard deviation in ABCA1 mRNA after treatment of 518A2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof ABCA1 mRNA in 518A2 cells are significantly increased 48 h aftertreatment with six of the oligos designed to ABCA1 antisense AK311445.Bars denoted as CUR-1214 to CUR-1222 correspond to samples treated withSEQ ID NOS: 26 to 34 respectively.

FIG. 1C is a graph of real time PCR results showing the foldchange+standard deviation in ABCA1 mRNA after treatment of 3T3 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof ABCA1 mRNA in 3T3 cells are significantly increased 48 h aftertreatment with three of the oligos designed to mouse ABCA1 antisenseBF133827. Bars denoted as CUR-1087 to CUR-1090, CUR-1092 and CUR-1091correspond to samples treated with SEQ ID NOS: 35 to 38, 40 and 39respectively.

FIG. 1D is a graph of real time PCR results showing the foldchange+standard deviation in LCAT mRNA after treatment of Hek293 cellswith siRNA and phosphorothioate oligonucleotides introduced usingLipofectamine 2000, as compared to control. Real time PCR results showthat the levels of the LCAT mRNA in Hek293 cells are significantlyincreased 48 h after treatment with three of the oligos designed to LCATantisense Hs.668679. Bats denoted as CUR-0476, CUR-0478. CUR-0822,CUR-0820 and CUR-0819 correspond to samples treated with SEQ ID NOS: 41,42, 58, 56 and 55 respectively.

FIG. 1E is a graph of real time PCR results showing the foldchanger-standard deviation in LCAT mRNA after treatment of HepG2 cellswith siRNA oligonucleotides introduced using Lipofectamine 2000, ascompared to control. Real time PCR results show that the levels of theLCAT mRNA in HepG2 cells are significantly increased 48 h aftertreatment with two of the oligos designed to LCAT antisense Hs.668679.Bars denoted as CUR-0476, CUR-0478, CUR-0444, CUR-0446, CUR-0448 andCUR-0450 correspond to samples treated with SEQ ID NOS: 41, 42, and 44to 47 respectively.

FIG. 1F is a graph of real time PCR results showing the foldchange+standard deviation in LCAT mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof the LCAT mRNA in HepG2 cells are significantly increased 48 h aftertreatment with one of the oligos designed to LCAT antisense Hs.668679.Bars denoted as CUR-0819, CUR-0818, CUR-0817, CUR-0816, CUR-0815,CUR-0820, CUR-0821 and CUR-0822 correspond to samples treated with SEQID NOS: 55, 54, 53, 52, 51 and 56 to 58 respectively.

FIG. 1G is a graph of real time PCR results showing the foldchange+standard deviation in LCAT mRNA after treatment of Veto cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof the LCAT mRNA in Veto cells are significantly increased 48 h aftertreatment with one of the oligos designed to LCAT antisense Hs.668679.Bats denoted as CUR-0819, CUR-0818, CUR-0817, CUR-0816, CUR-0815,CUR-0820, CUR-0821 and CUR-0822 correspond to samples treated with SEQID NOS: 55, 54, 53, 52, 51 and 56 to 58 respectively.

FIG. 1H is a graph of real time PCR results showing the foldchange+standard deviation in LRP1 mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real Time PCR results show that levels ofLRP1 mRNA in HepG2 cells are significantly increased 48 h aftertreatment with oligos to LRP1 antisense DC401271. Bars denoted asCUR-0767 to CUR-0769, CUR-0771, CUR-0770, CUR-0775, CUR-0773, CUR-0772and CUR-0774 correspond to samples treated with SEQ ID NOS; 59 to 61,63, 62, 67, 65, 64 and 66 respectively.

FIG. 1I is a graph of real time PCR results showing the foldchange-standard deviation in LRP1 mRNA after treatment of Vero cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real Time PCR results show that levels ofLRP1 mRNA in Vero cells are significantly increased 48 h after treatmentwith oligos to LRP1 antisense DC401271 and Hs.711951. Bars denoted asCUR-0768, CUR-0767, CUR-0774 and CUR-0772 correspond to samples treatedwith SEQ ID NOS: 60, 59, 66 and 64 respectively.

FIG. 1J is a graph of real time PCR results showing the foldchange+standard deviation in LRP1 mRNA after treatment of 3T3 cells withphosphorothioate oligonucleotides introduced using Lipofectamine 2000,as compared to control. Real Time PCR results show that levels of LRP1mRNA in 3T3 cells are significantly increased 48 h after treatment witholigos to LRP1 antisense DC401271 and AW544265. Bars denoted as CUR-1017to CUR-1022 correspond to samples treated with SEQ ID NOS: 68 to 73respectively.

FIG. 1K is a graph of real time PCR results showing the foldchange+standard deviation in LDLR mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real Time PCR results show that levels ofLDLR mRNA in HepG2 cells are significantly increased 48 h aftertreatment with antisense oligos to LDLR antisense sherflor.aApr07. Barsdenoted as CUR-1054 to CUR-1059 correspond to samples treated with SEQID NOS: 74 to 79 respectively.

FIG. 1L is a graph of real time PCR results showing the foldchange+standard deviation in LDLR mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real Time PCR results show that levels ofLDLR mRNA in HepG2 cells after treatment with antisense oligos to LDLRantisense bloflor.aApr07. Bars denoted as CUR-1059 to CUR-1063correspond to samples treated with SEQ ID NOS: 79 to 83 respectively.

FIG. 1M is a graph of real time PCR results showing the foldchange+standard deviation in ApoE mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof APOE mRNA in HepG2 cells are significantly increased 48 h aftertreatment with three of the antisense oligos designed to APOE antisenseHs.626623. Bars denoted as CUR-0978, CUR-0980, CUR-0981, CUR-0979,CUR-0973, CUR-0975, CUR-0974, CUR-0977 and CUR-0976 correspond tosamples treated with SEQ ID NOS: 89, 91, 92, 90, 84, 86, 85, 88 and 87respectively.

FIG. 1N to FIG. 1P represent a graphs of real time PCR results showingthe fold change-standard deviation in ApoA1 mRNA after treatment ofHepG2 cells with phosphorothioate oligonucleotides introduced usingLipofectamine 2000, as compared to control. Real time PCR results showthat the levels of ApoA1 mRNA in HepG2 cells are significantly increased48 h after treatment with some of the antisense oligonucleotides toApoA1 antisense DA327409ext. Bars RH3-RH597, correspond to samplestreated with SEQ ID NOS 171 to 248 respectively.

FIG. 1Q is a graph of real time PCR results showing the fold change inApoA1 mRNA (top panel) and ApoA1 natural antisense DA327409ext RNA(bottom panel) after treatment of HepG2 cells with naked LNA orphosphothioate oligonucleotides over 7 days as compared to control. Barsdenoted as #6LN A, #11 LNA, #6PS and #11PS represent SEQ ID NOS 249 to252 respectively.

FIG. 1R is a graph of real time PCR results showing the fold change inApoA1 mRNA (orange bats) and ApoA1 natural antisense DA327409ext RNA(bloc bars) after treatment of HepG2 cells with LNA oligonucleotides.Bars denoted as 6-11 correspond to SEQ ID NOS 249, 257 to 260 and 250.

FIG. 1S shows dose dependent increase in ApoA1 mRNA (bottom panel) andprotein (top panel) after treatment of HepG2 cells witholigonucleotides. Bars denoted CUR-4806 and CUR-4811 correspond to SEQID NOS 249 and 250 respectively.

FIG. 1T is a graph of the real time PCR results showing upregulation ofthe ApoA1 mRNA in primary African green monkey hepatocytes aftertreatment with oligonucleotides against natural ApoA1 antisenseDA327409ext. Bars denoted CUR-4816 and CUR-4811 correspond to SEQ IDNOS: 263 and 250 respectively.

FIG. 1U is a graph showing that ApoA1 mRNA and protein levels increasedin monkey liver biopsies after treatment with CUR-962, anoligonucleotide designed to ApoA1 antisense DA327409ext, compared to thebaseline levels, as determined by real time PCR and ELISA respectively(right panels). ApoA1 mRNA or protein levels did not change after thesame period of time in the control group dosed with an oligonucleotidethat showed no effect on ApoA1 levels in vitro (CUR-963) (left panels).Bars denoted CUR-962 and CUR-963 correspond to SEQ ID NOS 260 and 261respectively.

FIG. 2 shows

SEQ ID NO: 1: Homo sapiens ATP-binding cassette, sub-family A (ABC1)member 1 (ABCA1), mRNA (NCB1 Accession No.: NM.sub.--005502).

SEQ ID NO: 2: Homo sapiens lecithin-cholesterol acyltransferase (LCAT),mRNA (NCBI Accession No.: NM.sub.--000229.1).

SEQ ID NO: 3: Homo sapiens low density lipoprotein receptor-relatedprotein 1 (LRP1). mRNA (NCBI Accession No.: NM.sub.--002332.2).

SEQ ID NO: 4: Mus musculus low density lipoprotein receptor-relatedprotein 1 (Lrp1), mRNA (NCBI Accession No.: NM.sub.--008512.2).

SEQ ID NO: 5: Homo sapiens low density lipoprotein receptor (LDLR), mRNA(NCBI Accession No.: NM.sub.-000527.3).

SEQ ID NO: 6: Homo sapiens apolipoprotein E (APOE), mRNA (NCBI AccessionNo.: NM.sub.--000041.2).

SEQ ID NO: 7: Homo sapiens apolipoprotein A-I (APOA1), mRNA (NCBIAccession No.: NM.sub.--000039).

FIG. 3 shows

SEQ ID NO: 8: Human Natural ABCA1 antisense sequence (AK311445)

SEQ ID NO: 9: Mouse Natural ABCA1 antisense sequence (BF133827)

SEQ ID NO: 10: Human Natural LCAT antisense sequence (Hs.668679)

SEQ ID NO: 11: Human Natural LCAT antisense sequence (Hs.593769)

SEQ ID NO: 12: Human Natural LCAT antisense sequence (Hs.387239)

SEQ ID NO: 13: Human Natural LRP1 antisense sequence (Hs.711951)

SEQ ID NO: 14: Human Natural LRP1 antisense sequence (DC401271)

SEQ ID NO: 15: Human Natural LRP1 antisense sequence (BM933147)

SEQ ID NO: 16: Mouse Natural LRP1 antisense sequence (CK626173)

SEQ ID NO: 17: Mouse Natural LRP1 antisense sequence (AW544265) SEQ IDNO: 18: Human Natural ABCA1 antisense sequence (bloflor.aApr07)

SEQ ID NO: 19: Human Natural ABCA1 antisense sequence (sherflor.aApr07)

SEQ ID NO: 20: Natural APOE antisense sequence (Hs.626623)

SEQ ID NO: 21: Natural APOE antisense sequence (Hs.714236)

SEQ ID NO: 22: Natural APOA1 antisense sequence (DA327409 extended)

FIG. 4 shows the ABCA1 antisense oligonucleotides, SEQ ID NOs: 23 to40. * indicates phosphothioate bond.

FIG. 5 shows the LCAT antisense oligonucleotides, SEQ ID NOs: 41 to58. * indicates phosphothioate bond.

FIG. 6 shows the LRP1 antisense oligonucleotides, SEQ ID NOs: 59 to73. * indicates phosphothioate bond.

FIG. 7 shows the LDLR antisense oligonucleotides, SEQ ID NOs: 74 to83. * indicates phosphothioate bond.

FIG. 8 shows the ApoE antisense oligonucleotides, SEQ ID NOs: 84 to92. * indicates phosphothioate bond.

FIG. 9 shows the ApoA1 antisense oligonucleotides, SEQ ID NOs: 93 to263. * indicates phosphothioate bond.

FIG. 10 shows the ABCA1 sense oligonucleotides, SEQ ID NOs: 264 to 266.The sense oligonucleotides SEQ ID NOs: 264 to 266 are the reversecomplements of the antisense oligonucleotides SEQ ID NOs: 23 to 25respectively.

FIG. 11 shows the LCAT sense oligonucleotides, SEQ ID NOs: 267 to 274.The sense oligonucleotides SEQ ID NOs: 267 to 274 are the reversecomplements of the antisense oligonucleotides SEQ ID NOs: 41 to 48respectively.

FIG. 12 shows:

SEQ ID NOs: 275 to 277: correspond to the probe sequence, forward primersequence and the reverse primer sequence respectively with respect tothe custom designed assay for ApoA1 antisense DA327409ext

SEQ ID NO: 278: corresponds to CUR 962, * indicates phosphothioate bondand + indicates LNA.

SEQ ID NO: 279: corresponds to CUR 963, * indicates phosphothioate bondand + indicates LNA.

DETAILED DESCRIPTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a lull understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the ordering ofacts or events, as some acts may occur in different orders and/orconcurrently with other acts or events. Furthermore, not all illustratedacts or events are required to implement a methodology in accordancewith the present invention.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the genes disclosedherein, which in some embodiments relate to mammalian nucleic acid andamino acid sequences are intended to encompass homologous and/ororthologous genes and gene products from other animals including, butnot limited to other mammals, fish, amphibians, reptiles, and birds. Inpreferred embodiments, the genes or nucleic acid sequences are human.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As used herein, the term “mRNA” means the presently known mRNAtranscript(s) of a targeted gene, and any further transcripts which maybe elucidated.

By “antisense oligonucleotides” or “antisense compound” is meant an RNAor DNA molecule that binds to another RNA or DNA (target RNA, DNA). Forexample, if it is an RNA oligonucleotide it binds to another RNA targetby means of RNA-RNA interactions and alters the activity of the targetRNA (Eguchi et al., (1991) Ann. Rev. Biochem. 60, 631-652). An antisenseoligonucleotide can upregulate or downregulate expression and/orfunction of a particular polynucleotide. The definition is meant toinclude any foreign RNA or DNA molecule which is useful from atherapeutic, diagnostic, or other viewpoint. Such molecules include, forexample, antisense RNA or DNA molecules, interference RNA (RNAi), microRNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNAand agonist and antagonist RNA, antisense oligomeric compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, primers, probes, and otheroligomeric compounds that hybridize to al least a portion of the targetnucleic acid. As such, these compounds may be introduced in the form ofsingle-stranded, double-stranded, partially single-stranded, or circularoligomeric compounds.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. The term “oligonucleotide”, alsoincludes linear or circular oligomers of natural and/or modifiedmonomers or linkages, including deoxyribonucleosides, ribonucleosides,substituted and alpha-anomeric forms thereof, peptide nucleic acids(PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate,and the like. Oligonucleotides are capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, Hoogstecn orreverse Hogsteen types of base pairing, or the like.

The oligonucleotide may be “chimeric”, that is, composed of differentregions. In the context of this invention “chimeric” compounds areoligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one monomer unit, i.e., a nucleotide inthe case of an oligonucleotides compound. These oligonucleotidestypically comprise at least one region wherein the oligonucleotide ismodified in order to exhibit one or more desired properties. The desiredproperties of the oligonucleotide include, but are not limited, forexample, to increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. Different regions of the oligonucleotide may thereforehave different properties. The chimeric oligonucleotides of the presentinvention can be formed as mixed structures of two or moreoligonucleotides, modified oligonucleotides, oligonucleosides and/oroligonucleotide analogs as described above.

The oligonucleotide can be composed of regions that can be linked in“register” that is, when the monomers are linked consecutively, as innative DNA, or linked via spacers. The spacers are intended toconstitute a covalent “bridge” between the regions and have in preferredcases a length not exceeding about 100 carbon atoms. The spacers maycarry different functionalities, for example, having positive ornegative charge, carry special nucleic acid binding properties(intercalators, groove binders, toxins, fluorophors etc.), beinglipophilic, inducing special secondary structures like, for example,alanine containing peptides that induce alpha-helices.

As used herein “Lipid transport and metabolism genes” are inclusive ofall family members, mutants, alleles, fragments, species, coding andnoncoding sequences, sense and antisense polynucleotide strands, etc.

As used herein, the words ATP-binding cassette 1; Lipid transport andmetabolism gene; ABC transporter 1; cholesterol efflux regulatoryprotein (CERP), ABCA1, ABC-1, ABC1, CERP; FLJ14958; HDLDT1; TGD are usedinterchangeably in the present application.

As used herein, the words Lecithin-cholesterol acyltransferase, LCAT,Phosphatidylcholine-sterol acyltransferase, Phospholipid-cholesterolacyltransferase are used interchangeably in the present application.

As used herein, the words A2MR, Alpha-2-macroglobulin receptor, APOER,Apolipoprotein E receptor, APR, CD91, FLJ16451, IGFBP3R, LRP, LRP-1,MGC88725, Prolow-density lipoprotein receptor-related protein 1, TGFBR5are used interchangeably in the present application.

As used herein, the words LDLR, FH, FHC, LDLCQ2, LDL receptor,Low-density lipoprotein receptor are used interchangeably in the presentapplication.

As used herein, the words ADZ, Apo-E, ApoE, Apolipoprotein E, LDLCQ5,LPG, MGC1571 are used interchangeably in the present application.

As used herein, the words ApoA1, Apo-A1, Apolipoprotein A1, MGC117399are used interchangeably in the present application.

As used herein, the term “oligonucleotide specific for” or“oligonucleotide which targets” refers to an oligonucleotide having asequence (i) capable of forming a stable complex with a portion of thetargeted gene, or (ii) capable of forming a stable duplex with a portionof a mRNA transcript of the targeted gene. Stability of the complexesand duplexes can be determined by theoretical calculations and/or invitro assays. Exemplary assays for determining stability ofhybridization complexes and duplexes are described in the Examplesbelow.

As used herein, the term “target nucleic acid” encompasses DNA, RNA(comprising premRNA and mRNA) transcribed from such DNA and also cDNAderived from such RNA, coding, noncoding sequences, sense or antisensepolynucleotides. The specific hybridization of an oligomeric compoundwith its target nucleic acid interferes with the normal function of thenucleic acid. This modulation of function of a target nucleic acid bycompounds, which specifically hybridize to it, is generally referred toas “antisense” The functions of DNA to be interfered include, forexample, replication and transcription. The functions of RNA to beinterfered, include all vital functions such as, for example,translocation of the RNA to the she of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in or facilitatedby the RNA. The overall effect of such interference with target nucleicacid function is modulation of the expression of an encoded product oroligonucleotides.

RNA interference “RNAi” is mediated by double stranded RNA (dsRNA)molecules that have sequence-specific homology to their “target” nucleicacid sequences (Caplen, N.J., et al. (2001) Proc. Natl. Acad. Sci. USA98:9742-9747). In certain embodiments of the present invention, themediators are 5-25 nucleotide “small interfering” RNA duplexes (siRNAs).The siRNAs are derived from the processing of dsRNA by an RNase enzymeknown as Dicer (Bernstein, E., et al. (2001) Nature 409:363-366). siRNAduplex products are recruited into a multi-protein siRNA complex termedRISC (RNA Induced Silencing Complex). Without wishing to be bound by anyparticular theory, a RISC is then believed to be guided to a targetnucleic acid (suitably mRNA), where the siRNA duplex interacts in asequence-specific way to mediate cleavage in a catalytic fashion(Bernstein, E., et al. (2001) Nature 409:363-366; Boutla, A., et al.(2001) Curr Biol. 11:1776-1780). Small interfering RNAs that can be usedin accordance with the present invention can be synthesized and usedaccording to procedures that are well known in the art and that will befamiliar to the ordinarily skilled artisan. Small interfering RNAs foruse in the methods of the present invention suitably comprise betweenabout 1 to about 50 nucleotides (nt). In examples of non limitingembodiments, siRNAs can comprise about 5 to about 40 nt, about 5 toabout 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about20-25 nucleotides.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the ease of genes that have not been sequenced,Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

By “enzymatic RNA” is meant an RNA molecule with enzymatic activity(Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymaticnucleic acids (ribozymes) act by first binding to a target RNA. Suchbinding occurs through the target binding portion of an enzymaticnucleic acid which is held in close proximity to an enzymatic portion ofthe molecule that acts to cleave the target RNA. Thus, the enzymaticnucleic acid first recognizes and then binds a target RNA through basepairing, and once bound to the correct site, acts enzymatically to cutthe target RNA.

By “decoy RNA” is meant an RNA molecule that mimics the natural bindingdomain for a ligand. The decoy RNA therefore competes with naturalbinding target for the binding of a specific ligand. For example, it hasbeen shown that over-expression of HIV trans-activation response (TAR)RNA can act as a “decoy” and efficiently binds HIV tat protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA(Sullenger et al. (1990) Cell, 63, 601-608). This is meant to be aspecific example. Those in the art will recognize that this is but oneexample, and other embodiments can be readily generated using techniquesgenerally known in the art.

As used herein, the term “monomers” typically indicates monomers linkedby phosphodiester bonds or analogs thereof to form oligonucleotidesranging in size from a few monomeric units, e.g., from about 3-4, toabout several hundreds of monomeric units. Analogs of phosphodiesterlinkages include: phosphorothioate, phosphorodithioate,methylphosphornates, phosphoroselenoate, phosphoramidate, and the like,as more fully described below.

The term “nucleotide” covers naturally occurring nucleotides as well asnonnaturally occurring nucleotides. It should be clear to the personskilled in the art that various nucleotides which previously have beenconsidered “non-naturally occurring” have subsequently been found innature. Thus, “nucleotides” includes not only the known purine andpyrimidine heterocycles-containing molecules, but also heterocyclicanalogues and tautomers thereof. Illustrative examples of other types ofnucleotides are molecules containing adenine, guanine, thymine,cytosine, uracil, purine, xanthine, diaminopurine,8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine,N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine and the “non-naturally occurring” nucleotidesdescribed in Benner et at, U.S. Pat. No. 5,432,272. The term“nucleotide” is intended to cover every and all of these examples aswell as analogues and tautomers thereof. Especially interestingnucleotides are those containing adenine, guanine, thymine, cytosine,and uracil, which are considered as the naturally occurring nucleotidesin relation to therapeutic and diagnostic application in humans.Nucleotides include the natural 2′-deoxy and 2′-hydroxyl sugars, e.g.,as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman,San Francisco, 1992) as well as their analogs.

“Analogs” in reference to nucleotides includes synthetic nucleotideshaving modified base moieties and/or modified sugar moieties (sec e.g.,described generally by Scheit, Nucleotide Analogs, John Wiley, New York,1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443,Toulme, J. J., (2001) Nature Biotechnology 19:17-18; Manoharan M.,(1999) Biochemica et Biophysica Acta 1489:117-139; Freier S. M., (1997)Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery &Development, 3: 203-213, Herdewin P., (2000) Antisense & Nucleic AcidDrug Dev., 10:297-310); 2′-0, 3′-C-linked [3.2.0]bicycloarabinonucleosides (see e.g. N. K Christiensen, et al, (1998) J.Am. Chem. Soc., 120: 5458-5463; Prakash T P, Bhat B. (2007) Curr Top MedChem. 7(7):641-9; Cho E J, et al. (2009) Annual Review of AnalyticalChemistry, 2, 241-264). Such analogs include synthetic nucleotidesdesigned to enhance binding properties, e.g., duplex or triplexstability, specificity, or the like.

As used herein, “hybridization” means the pairing of substantiallycomplementary strands of oligomeric compounds. One mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleotides) of the strands of oligomeric compounds.For example, adenine and thymine are complementary nucleotides whichpair through the formation of hydrogen bonds. Hybridization can occurunder varying circumstances.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal (unctionof the target nucleic acid to cause a modulation of (unction and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the ease of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated. In general, stringent hybridization conditionscomprise low concentrations (<0.15M) of salts with inorganic cationssuch as Na++ or K++ (i.e., low ionic strength), temperature higher than20.degree. C.-25.degree. C. below the Tm of the oligomericcompound:target sequence complex, and the presence of denaturants suchas formamide, dimethylformamide, dimethyl sulfoxide, or the detergentsodium dodecyl sulfate (SDS). For example, the hybridization ratedecreases 1.1% for each 1% formamide. An example of a high stringencyhybridization condition is 0.1.times, sodium chloride-sodium citratebuffer (SSC)/0.1% (w/v) SDS at 60.degree. C. for 30 minutes.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides on one or two oligomeric strands. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, said target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleotides such that stable and specificbinding occurs between the oligomeric compound and a target nucleicacid.

It is understood in the art dial the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligomeric compounds of the presentinvention comprise at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an antisense compound in which 18 of 20nucleotides of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remaining noncomplementarynucleotides may be clustered or interspersed with complementarynucleotides and need not be contiguous to each other or to complementarynucleotides. As such, an antisense compound which is 18 nucleotides inlength having 4 (four) noncomplementary nucleotides which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., (1990) J. Mol. Biol., 215, 403-410; Zhang and Madden,Genome Res., 7, 649-656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., (1981) 2, 482-489).

As used herein, the term “Thermal Melting Point (Tm)” refers to thetemperature, under defined ionic strength, pH, and nucleic acidconcentration, at which 50% of the oligonucleotides complementary to thetarget sequence hybridize to the target sequence at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is at least about 0.01 to 1.0 M Na ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about30.degree. C. for short oligonucleotides (e.g., 10 to 50 nucleotide).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide.

As used herein, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene.

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype gene products. Variants may result from at least one mutation inthe nucleic acid sequence and may result in altered mRNAs or inpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility versus resistance.

Derivative polynucleotides include nucleic acids subjected to chemicalmodification, for example, replacement of hydrogen by an alkyl, acyl, oramino group. Derivatives, e.g., derivative oligonucleotides, maycomprise non-naturally-occurring portions, such as altered sugarmoieties or inter-sugar linkages. Exemplary among these arephosphorothioate and other sulfur containing species which are known inthe art. Derivative nucleic acids may also contain labels, includingradionucleotides, enzymes, fluorescent agents, chemiluminescent agents,chromogenic agents, substrates, cofactors, inhibitors, magneticparticles, and the like.

A “derivative” polypeptide or peptide is one that is modified, forexample, by glycosylation, pegylation, phosphorylation, sulfation,reduction/alkylation, acylation, chemical coupling, or mild formalintreatment. A derivative may also be modified to contain a detectablelabel, either directly or indirectly, including, but not limited to, aradioisotope, fluorescent, and enzyme label.

As used herein, the term “animal” or “patient” is meant to include, forexample, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys,horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken,reptiles, fish, insects and arachnids.

“Mammal” covers warm blooded mammals that are typically under medicalcare (e.g., humans and domesticated animals). Examples include feline,canine, equine, bovine, and human, as well as just human.

“Treating” or “treatment” covers the treatment of a disease-state in amammal, and includes: (a) preventing the disease-state from occurring ina mammal, in particular, when such mammal is predisposed to thedisease-state but has not yet been diagnosed as having it; (b)inhibiting the disease-state, e.g., arresting it development; and/or (c)relieving the disease-state, e.g., causing regression of the diseasestate until a desired endpoint is reached. Treating also includes theamelioration of a symptom of a disease (e.g., lessen the pain ordiscomfort), wherein such amelioration may or may not be directlyaffecting the disease (e.g., cause, transmission, expression, etc.).

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in mammals, including, but not limited to:leukemias, lymphomas, melanomas, carcinomas and sarcomas. The cancermanifests itself as a “tumor” or tissue comprising malignant cells ofthe cancer. Examples of tumors include sarcomas and carcinomas such as,but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Additional cancers which can be treated by the disclosedcomposition according to the invention include but not limited to, forexample, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,adrenal cortical cancer, and prostate cancer.

“Neurological disease or disorder” refers to any disease or disorder ofthe nervous system and/or visual system. “Neurological disease ordisorder” include disease or disorders that involve the central nervoussystem (brain, brainstem and cerebellum), the peripheral nervous system(including cranial nerves), and the autonomic nervous system (parts ofwhich are located in both central and peripheral nervous system).Examples of neurological disorders include but are not limited to,headache, stupor and coma, dementia, seizure, sleep disorders, trauma,infections, neoplasms, neuroopthalmology, movement disorders,demyelinating diseases, spinal cord disorders, and disorders ofperipheral nerves, muscle and neuromuscular junctions. Addiction andmental illness, include, but are not limited to, bipolar disorder andschizophrenia, are also included in the definition of neurologicaldisorder. The following is a list of several neurological disorders,symptoms, signs and syndromes that can be treated using compositions andmethods according to the present invention: acquired epileptiformaphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy;age-related macular degeneration; agenesis of the corpus callosum;agnosia; Aicardi syndrome; Alexander disease; Alpers' disease;alternating hemiplegia; Vascular dementia; amyotrophic lateralsclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia;aphasia; apraxia; arachnoid cysts; arachnoiditis; Anronl-Chiarimalformation; arteriovenous malformation; Asperger syndrome; ataxiatelegiectasia; attention deficit hyperactivity disorder, autism;autonomic dysfunction; back pain; Batten disease; Behcet's disease;Bell's palsy; benign essential blepharospasm; benign focal; amyotrophy;benign intracranial hypertension; Binswanger's disease; blepharospasm;Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; braininjury; brain tumors (inducting glioblastoma multiforme); spinal tumor;Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome;causalgia; central pain syndrome; central pontine myelinolysis; cephalicdisorder, cerebral aneurysm; cerebral arteriosclerosis; cerebralatrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Toothdisease; chemotherapy-induced neuropathy and neuropathic pain; Chiarimalformation; chorea; chronic inflammatory demyelinating polyneuropathy;chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome;coma, including persistent vegetative state; congenital facial diplegia;corticobasal degeneration; cranial arteritis; craniosynostosis;Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing'ssyndrome; cytomegalic inclusion body disease; cytomegalovirus infection;dancing eyes-dancing feet syndrome; DandyWalker syndrome; Dawsondisease; De Morsier's syndrome; Dejerine-Klumke palsy; dementia;dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia;dysgraphia; dyslexia; dystonias; early infantile epilepticencephalopathy; empty sella syndrome; encephalitis; encephaloceles;encephalotrigeminal angiomatosis; epilepsy, Erb's palsy; essentialtremor; Fabry's disease; Fahr's syndrome; feinting; familial spasticparalysis; febrile seizures; Fisher syndrome; Friedreich's ataxia;fronto-temporal dementia and other “tauopathies”; Gaucher's disease;Gerstmann's syndrome; giant cell arteritis; giant cell inclusiondisease; globoid cell leukodystrophy; Guillain-Barre syndrome;HTLV-1-associated myelopathy; Hallervorden-Spatz disease; head injury;headache; hemifacial spasm; hereditary spastic paraplegia; heredopathiaatactic a polyneuritiformis; herpes zoster oticus; herpes zoster.Hirayama syndrome; HIV associated dementia and neuropathy (alsoneurological manifestations of AIDS); holoprosencephaly, Huntington'sdisease and other polyglutamine repeat diseases; hydranencephaly;hydrocephalus; hypercortisolism; hypoxia; immune-mediatedencephalomyelitis; inclusion body myositis; incontinentia pigmenti;infantile phytanic acid storage disease; infantile refsum disease;infantile spasms; inflammatory myopathy; intracranial cyst; intracranialhypertension; Joubert syndrome; Keams-Sayre syndrome; Kennedy diseaseKinsboume syndrome; Klippel Feil syndrome; Krabbe disease;Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eatonmyasthenic syndrome; Landau-Kleffner syndrome; lateral medullary(Wallenberg) syndrome; learning disabilities; Leigh's disease;Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy bodydementia; Lissencephaly; locked-in syndrome; Lou Gehrig's disease (i.e.,motor neuron disease or amyotrophic lateral sclerosis); lumbar discdisease; Lyme disease-neurological sequelae; Machado-Joscph disease;macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieresdisease; meningitis; Menkes disease; metachromatic leukodystrophy;microcephaly; migraine; Miller Fisher syndrome; mini-strokes;mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motorneuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infarctdementia; multifocal motor neuropathy; multiple sclerosis and otherdemyelinating disorders; multiple system atrophy with posturalhypotension; p muscular dystrophy; myasthenia gravis; myclinoclasticdiffuse sclerosis; myoclonic encephalopathy of infants; myoclonus;myopathy; myotonia congenital; narcolepsy; neurofibromatosis;neuroleptic malignant syndrome; neurological manifestations of AIDS;neurological sequelae of lupus; neuromyotonia; neuronal ceroidlipofuscinosis; neuronal migration disorders; Niemann-Pick disease;O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinaldysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy;opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overusesyndrome; paresthesia; Neurodegenerative disease or disorder(Parkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis andother diseases and disorders associated with neuronal cell death);paramyotonia congenital; paraneoplastic diseases; paroxysmal attacks;Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodicparalyses; peripheral neuropathy, painful neuropathy and neuropathicpain; persistent vegetative state; pervasive developmental disorders;photic sneeze reflex; phytanic acid storage disease; Pick's disease;pinched nerve; pituitary tumors; polymyositis; porencephaly; post-poliosyndrome; postherpetic neuralgia; postinfectious encephalomyelitis;postural hypotension; Prader-Willi syndrome; primary lateral sclerosis;prion diseases; progressive hemifacial atrophy; progressivemultifocalleukoencephalopathy; progressive sclerosing poliodystrophy;progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Huntsyndrome (types I and II); Rasmussen's encephalitis; reflex sympatheticdystrophy syndrome; Refsum disease; repetitive motion disorders;repetitive stress injuries; restless legs syndrome;retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; SaintVitus dance; Sandhoff disease; Schilder's disease; schizencephaly;septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Dragersyndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome; spasticity;spina bifida; spinal cord injury; spinal cord tumors; spinal muscularatrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome; subacutesclerosing panencephalitis; subcortical arteriosclerotic encephalopathy,Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachsdisease; temporal arteritis; tethered spinal cord syndrome; Thomsendisease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis;Tourette syndrome; transient ischemic attack; transmissible spongiformencephalopathies; transverse myelitis; traumatic brain injury; tremor;trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis;vascular dementia (multi-infarct dementia); vasculitis includingtemporal arteritis; Von Hippel-Lindau disease; Wallenberg's syndrome;Werdnig-Hofifman disease; West syndrome; whiplash; Williams syndrome;Wildon's disease; and Zellweger syndrome.

An “Inflammation” refers to systemic inflammatory conditions andconditions associated locally with migration and attraction ofmonocytes, leukocytes and/or neutrophils. Examples of inflammationinclude, but are not limited to, Inflammation resulting from infectionwith pathogenic organisms (including gram-positive bacteria,gram-negative bacteria, viruses, fungi, and parasites such as protozoaand helminths), transplant rejection (including rejection of solidorgans such as kidney, liver, heart, lung or cornea, as well asrejection of bone marrow transplants including graft-versus-host disease(GVHD)), or from localized chronic or acute autoimmune or allergicreactions. Autoimmune diseases include acute glomerulonephritis;rheumatoid or reactive arthritis; chronic glomerulonephritis;inflammatory bowel diseases such as Crohn's disease, ulcerative colitisand necrotizing enterocolitis; granulocyte transfusion associatedsyndromes; inflammatory dermatoses such as contact dermatitis, atopicdermatitis, psoriasis; systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, and some forms of diabetes, or anyother autoimmune state where attack by the subject's own immune systemresults in pathologic tissue destruction. Allergic reactions includeallergic asthma, chronic bronchitis, acute and delayed hypersensitivity.Systemic inflammatory disease states include inflammation associatedwith trauma, burns, reperfusion following ischemic events (e.g.thrombotic events in heart, brain, intestines or peripheral vasculature,including myocardial infarction and stroke), sepsis, ARDS or multipleorgan dysfunction syndrome. Inflammatory cell recruitment also occurs inatherosclerotic plaques. Inflammation includes, but is not limited to,Non-Hodgkins lymphoma, Wegener's granulomatosis, Hashimoto'sthyroiditis, hepatocellular carcinoma, thymus atrophy, chronicpancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia,osteoarthritis, ulcerative colitis, papillary carcinoma, Crohn'sdisease, ulcerative colitis, acute cholecystitis, chronic cholecystitis,cirrhosis, chronic sialadenitis, peritonitis, acute pancreatitis,chronic pancreatitis, chronic Gastritis, adenomyosis, endometriosis,acute cervicitis, chronic cervicitis, lymphoid hyperplasia, multiplesclerosis, hypertrophy secondary to idiopathic thrombocytopenic purpura,primary IgA nephropathy, systemic lupus erythematosus, psoriasis,pulmonary emphysema, chronic pyelonephritis, and chronic cystitis.

A cardiovascular disease or disorder includes those disorders that caneither cause ischemia or are caused by reperfusion of the heart.Examples include, but are not limited to, atherosclerosis, coronaryartery disease, granulomatous myocarditis, chronic myocarditis(non-granulomatous), primary hypertrophic cardiomyopathy, peripheralartery disease (PAD), stroke, angina pectoris, myocardial infarction,cardiovascular tissue damage caused by cardiac arrest, cardiovasculartissue damage caused by cardiac bypass, cardiogenic shock, and relatedconditions that would be known by those of ordinary skill in the art orwhich involve dysfunction of or tissue damage to the heart orvasculature, especially, but not limited to, tissue damage related to aLipid transport and metabolism gene activation. CVS diseases include,but are not limited to, atherosclerosis, granulomatous myocarditis,myocardial infarction, myocardial fibrosis secondary to valvular heartdisease, myocardial fibrosis without infarction, primary hypertrophiccardiomyopathy, and chronic myocarditis (non-granulomatous).

A ‘Metabolic disease or disorder’ refers to a wide range of diseases anddisorders of the endocrine system including, for example, insulinresistance, diabetes, obesity, impaired glucose tolerance, high bloodcholesterol, hyperglycemia, hyperinsulinemia, dyslipidemia andhyperlipidemia.

Polynucleotide and Oligonucleotide Compositions and Molecules

Targets

In one embodiment, the targets comprise nucleic acid sequences of aLipid transport and metabolism genes, including without limitation senseand/or antisense noncoding and/or coding sequences associated with aLipid transport and metabolism gene.

In one embodiment, the targets comprise nucleic acid sequences of ABCA1,including without limitation sense and/or antisense noncoding and/orcoding sequences associated with ABC A1 gene.

In one embodiment, the targets comprise nucleic acid sequences of LCAT,including without limitation sense and/or antisense noncoding and/orcoding sequences associated with LCAT gene.

In one embodiment, the targets comprise nucleic acid sequences of LRP1,including without limitation sense and/or antisense noncoding and/orcoding sequences associated with LRP1 gene.

In one embodiment, the targets comprise nucleic acid sequences of lowdensity lipoprotein receptor (LDLR), including without limitation senseand/or antisense noncoding and/or coding sequences associated with LDLR.

In one embodiment, the targets comprise nucleic acid sequences ofapolipoprotein (ApoA1), including without limitation sense and/orantisense noncoding and/or coding sequences associated with ApoA. Humanapolipoprotein A-I (ApoA-I) is the major protein constituent ofhigh-density lipoproteins (HDL and lymph chylomicrons. In human plasmafour major circulating lipoproteins have been named: chylomicrons (CM),very low-density lipoproteins (VLDL), low-density lipoproteins (LDL),and high-density lipoproteins (HDL). HDL is involved in the removal ofcholesterol from peripheral tissues by transporting it to the liver orto other lipoproteins.

ATP-binding cassette, sub family-A (ABCAI) member I ABCAI functions as acholesterol efflux pump in the cellular lipid removal pathway.

ATP-binding cassette transporters (ABC-transporter) are members of aprotein superfamily that is one of the largest and most ancient familieswith representatives in all extant phyla from prokaryotes to humans. ABCtransporters are transmembrane proteins that utilize the energy ofadenosine triphosphate (ATP) hydrolysis to carry out certain biologicalprocesses including translocation of various substrates across membranesand non-transport-related processes such as translation of RNA and DNArepair. They transport a wide variety of substrates across extra- andintracellular membranes, including metabolic products, lipids andsterols, and drugs. Proteins are classified as ABC transporters based onthe sequence and organization of their ATP-binding cassette (ABC)domain(s). ABC transporters are involved in tumor resistance, cysticfibrosis, bacterial multidrug resistance, and a range of other inheritedhuman diseases.

The membrane-associated protein encoded by ABC1 gene is a member of thesuperfamily of ATP-binding cassette (ABC) transporters. ABC proteinstransport various molecules across extra- and intracellular membranes.ABC genes are divided into seven distinct subfamilies (ABCA, MDRITAP,MRP, ALD, OABP, GCN20, White). This protein is a member of the ABCAsubfamily. Members of the ABCA subfamily comprise the only major ABCsubfamily found exclusively in multicellular eukaryotes. Withcholesterol as its substrate, this protein functions as a cholesterolefflux pump in the cellular lipid removal pathway.

In preferred embodiments, antisense oligonucleotides are used to preventor treat diseases or disorders associated with ATP-Binding cassettemolecules. ATP-binding cassette transporter ABC1 (member 1 of humantransporter sub-family ABCA), also known as the cholesterol effluxregulatory protein (CERP) is a protein which in humans is encoded by theABC1 gene. This transporter is a major regulator of cellular cholesteroland phospholipid homeostasis.

ABC1 is present in high-density lipoproteins (HDL) which permits theremoval of excessive cholesterol and phospholipids from human cellmembranes. Since this protein is needed throughout the body it issynthesis ubiquitously as a 220-kDa protein. It is present in higherquantities in tissues that shuttle or are involved in the turnover oflipids such as the liver, the small intestine and adipose tissue.

Factors that act upon the ABC1 transporter's expression or itsposttranslational modification are also molecules that are involved inits subsequent function like fatty acids, cholesterol and also cytokinesand cyclic adenosine monophosphate.

Low-density lipoprotein receptor-related protein 1 (LPR1) is about 4544amino acids; 504575 Da. It is a heterodimer of an 85-kDa membrane-boundcarboxyl subunit and a noncovalently attached 515-kDa amino-terminalsubunit. Intracellular domain interacts with MAFB. LPR1 is found in acomplex with PIDIIPCLI1, LRP1 and CUBNI. Interacts with SNX17,PIDIIPCLI1, PDGF, LRPAP1 and CUBN. The intracellular domain interactswith SHC1, GULPI and DAB1.

LRP1 is an endocytic receptor involved in endocytosis and inphagocytosis of apoptotic cells; early embryonic development; cellularlipid homeostasis; plasma clearance of chylomicron remnants andactivated LRPAP1 (alpha 2-macroglobulin); local metabolism of complexesbetween plasminogen activators and their endogenous inhibitors. Withoutwishing to be bound by theory, it may modulate cellular events, such asAPP metabolism, kinase-dependent intracellular signaling, neuronalcalcium signaling as well as neurotransmission.

High density lipoprotein (HDL) picks up extra cholesterol in the bloodand returns it to the liver Low density lipoprotein (or LDL) is the maintransporter of cholesterol in the body. But too much LDL over many yearscan result in atherosclerosis (the narrowing and hardening of arteries)and lead to heart disease or a heart attack. The ratio is determined bydividing the LDL cholesterol into the HDL cholesterol. For example, if aperson has an HDL cholesterol of 50 mg/dL and an LDL cholesterol of 150mg/dL, the HDL/LDL ratio would be 0.33. The goal is to keep the HDL/LDLratio above 0.3, with the ideal HDL/LDL ratio being above 0.4.

HDL are synthesized de novo in both the liver and small intestine asprotein-rich disc-shaped particles. The primary apoproteins of HDL areapoA-1, apoA-II, apoC-I, apoC-II, and apoE. Newly formed HDL containvery little cholesterol and cholesteryl esters. HDL are converted fromtheir initial discoidal shape into spherical lipoprotein particlesthrough the accumulation of cholesteryl esters in the neutral core ofthe lipoprotein particle. Cholesterol is accumulated by HDL fromchylomicron remnants VLDL remnants (also called intermediate densityLipoproteins or IDL) and directly from cell surface membranes. Thecholesterol is esterified through the action of an HDL-associated enzymelecithin:cholesterol acyltransferase (“LCAT”). For LCAT to transfer afatty acid from lecithin (phosphatidylcholine) to the C-3-OH group ofcholesterol, interaction with ApoA-I found on the HDL surface isrequired. This accumulation of core cholesteryl esters converts nascentHDL to HDL2 and HDL3. Sec R. I. Levy et al., “The structure, functionand metabolism of high-density lipoproteins: A status report,”Circulation, vol. 62, pp. IV4-8 (1980); and D. I. Silverman et al.,“High-density lipoprotein subfractions,” Am. J. Med., vol. 94, pp.636-45 (1993).

HDL are usually isolated from the plasma by ultracentrifugation. Thenormal HDL density range is from 1.063 g/mL to 1.21 g/mL, which dividesroughly into two ranges HDL2 (1.063 g/mL to 1.125 g/mL) and HDL3 (1.125g/mL to 1.21 g/mL). More recently, two major populations of particles inHDL have been identified by two dimensional electrophoresis followed byimmunoblotting and enzyme-linked differential antibody immunosorbentassay. One of these populations contains particles with apoA-I alone,and the other contains particles with both apoA-I and apoA-II. Therelative proportion of apoA-I particles is highest in the HDL2 fraction,while HDL3 is more a combination of apoA-I and apoA-II. See J. C.Fruchart et al., “Apolipoprotein A-containing lipoprotein particles:physiological role, quantification, and clinical significance,” Clin.Chem., vol. 38, pp. 793-7 (1992); and B. F. Asztalos et al.,“Normolipidemic subjects with low HDL cholesterol levels have alteredHDL subpopulations,” Arteriosder. Thromb. Vase. Biol., vol. 17, pp.1885-1893 (1997).

Human apolipoprotein A-I (ApoA-I) is the major protein constituent ofHDL and lymph chylomicrons. ApoA-I is primarily synthesized in the liverand small intestine as a precursor protein (preproapo A-I). PreproapoA-I is cleaved intracellularly to form proapo A-I, the form secretedinto the plasma and lymph. In the plasma, six amino acids are cleavedfrom proapo A-I to form mature ApoA-I.

Mature ApoA-I is a single unglycosylated polypeptide composed of 243amino acids of known sequence. ApoA-I serves as a cofactor of a plasmaenzyme (lecithin-cholesterol acyltransferase (LCAT)), responsible forthe formation of most cholesterol esters in plasma. Decreased levels ofApoA-I may result in disorders of the plasma lipid transport system andin the development of coronary heart disease. Low levels of both ApoA-Iand HDL has been shown to be a strong risk factor for heart attacks andother atherosclerotic vascular diseases. Sec U.S. Pat. Nos. 5,059,528and 6,258,596.

Apolipoprotein E (ApoE) is an apoprotein found in the chylomicron andintermediate-density lipoproteins (IDLs) that binds to a specificreceptor cm liver cells and peripheral cells. Intermediate-densitylipoproteins belong to the lipoprotein particle family and are formedfrom the degradation of very low-density lipoproteins. IDL is one of thefive major groups of lipoproteins (chylomicrons, VLDL, IDL, LDL, HDL)that enable fats and cholesterol to move within the water-based solutionof the bloodstream. Apolipoprotein E (ApoE) is important for the normalcatabolism of triglyceride-rich lipoprotein constituents.

The APOE gene, ApoE, is mapped to chromosome 19 in a cluster withApolipoprotein C1 and Apolipoprotein C2. ApoE consists of four exons andthree introns, totaling 3597 base pairs. In melanocytic cells APOE geneexpression may be regulated by microphthalmia-associated transcriptionfactor (MITF). The gene is polymorphic with three major alleles, ApoE2,ApoE3, ApoE4, which translate into three isoforms of the protein:normal-ApoE-E3; dysfunctional-ApoE-E2 and ApoE-E4. These iso formsdiffer from each other only by single amino acid substitutions atpositions 112 and 158.

Lecithin-cholesterol acyltransferase (LCAT), is a plasma enzyme producedby the liver and catalyzes the conversion of cholesterol to cholesterylesters on lipoproteins by the transacylation of fatty acid from the sn-2position of phosphatidylcholine to the 3-hydroxyl group on the A-ring ofcholesterol. Most LCAT activity is found on high-density lipoprotein(HDL) but approximately 30% is also on apolipoprotein (Apo) B-containinglipoproteins.

The apolipoprotein E gene is polymorphic with three major alleles,ApoE2, ApoE3, ApoE4. E2 is associated with the genetic disorder type IIIhyperlipoproteinemia and with both increased and decreased risk foratherosclerosis. E3 is found in approximately 64 percent of thepopulation. It is considered the “neutral” Apo E genotype. E4 maycontribute to atherosclerosis and Alzheimer's disease, impairedcognitive function, and reduced neurite outgrowth.

LCAT promotes the reverse cholesterol transport pathway, the pathway bywhich excess cellular cholesterol is returned to the liver forexcretion. Without wishing to be bound by theory, mechanisms include,for example: LCAT increases the level of HDL, which in itself mayincrease the flux of cholesterol from cells by increasing the amount ofextracellular acceptors of cholesterol. Also, esterification ofcholesterol by LCAT on HDL could limit the spontaneous back exchange ofcholesterol from HDL to cells and promotes the net delivery ofcholesterol on HDL and on to the liver.

In preferred embodiments, antisense oligonucleotides are used to preventor treat diseases or disorders associated with Lipid transport andmetabolism gene family members. Exemplary Lipid transport and metabolismgene mediated diseases and disorders which can be treated withcell/tissues regenerated from stem cells obtained using the antisensecompounds comprise: a cardiovascular disease or disorder, a metabolicdisease or disorder (e.g., diabetes, obesity, dyslipidemia,hyperglycemia, hyperinsulinemia, hypercholesterolemia etc.), a diseaseor disorder associated with impaired lipid metabolism, a coronary arterydisease, atherosclerosis, an HDL metabolism disease or disorder (e.g.,familial HDL deficiency (FHD), Sea-blue histiocytosis, Tangier'sDisease, Fish-eye disease, LCAT deficiency, low-HDL cholesterolemiaetc.), a disease or disorder associated with cellular cholesterol and/orphospholipid homeostasis, Familial amyloid nephropathy, a disease ordisorder associated with impaired cholesterol regulation, a disease ordisorder associated with a deficiency of the Lipid transport andmetabolism gene transporter, Apolipoprotein A-I deficiency, a disease ordisorder associated with abnormally fast or abnormally stow rate ofcholesterol efflux in a cell, a disease or disorder associated withpancreatic beta cell function, diabetes, a metabolic disease ordisorder, arthritis, inflammation, an autoimmune disease or disorder,acquired immune deficiency syndrome (AIDS), inflammation, a neurologicaldisease or disorder, a neurodegenerative disease or disorder, cancer,dyslipidemia, metabolic syndrome, a senile plaque, cerebral amyloidangiopathy, Amyloidosis, glioblastoma, a disease or disorder associatedwith amyloid deposition, neurofibrillary tangles, choriocarcinoma,astrocytoma, amyloidosis, hyperlipidemia, neoplastic transformation,atherosclerotic plaque, obstruction, metastasis, pulmonary fibrosis,necrosis, shock, melanoma, genetic susceptibility, psoriasis, glioma,neuropathology, a vascular disease, cell damage. Nonsmall cell lungcarcinomas (NSCLCs), liposarcoma, an immunodeficiency disease ordisorder, an organ transplant rejection, an allergy, glomerulonephritis,venous thrombosis, pathological processes or leukemia, a skeletaldisease or disorder, a muscular disease or disorder, a disease ordisorder associated with infectious organisms, an immune related diseaseor disorder, nerve repair and paralysis, neuroendocrine differentiation,systemic non-neuropathic amyloidosis, an amyloid disease, tumor growthdependent on angiogenesis, non-cancerous diseases with symptoms includean increase in angiogenesis, e.g., psoriasis, retinopathy ofprematurity, a Choroid disease, neovascular glaucoma, diabeticretinopathy, substance abuse, impaired cognitive function, and reducedneurite outgrowth, ApoE abnormal expression, function, activity ascompared to a normal control, psoriasis, a disease or disorder caused byforeign organisms such as viral, bacterial, parasitic, fungal, and thelike.

In a preferred embodiment the Lipid transport and metabolism geneantisense oligonucleotides are therapeutically used in organtransplantation (e.g., liver transplant, kidney transplant, bone marrowtransplant, heart transplant etc.).

In a preferred embodiment, the oligonucleotides are specific forpolynucleotides of a Lipid transport and metabolism gene, whichincludes, without limitation noncoding regions. The Lipid transport andmetabolism gene targets comprise variants of a Lipid transport andmetabolism gene; mutants of a Lipid transport and metabolism gene,including SNPs; noncoding sequences of a Lipid transport and metabolismgene; alleles, fragments and the like. Preferably the oligonucleotide isan antisense RNA molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to a Lipid transport and metabolism genepolynucleotides alone but extends to any of the isoforms, receptors,homologs, non-coding regions and the like of a Lipid transport andmetabolism gene.

In another preferred embodiment, an oligonucleotide targets a naturalantisense sequence (natural antisense to the coding and non-codingregions) of a Lipid transport and metabolism gene targets, including,without limitation, variants, alleles, homologs, mutants, derivatives,fragments and complementary sequences thereto. Preferably theoligonucleotide is an antisense RNA or DNA molecule.

In another preferred embodiment, the oligomeric compounds of the presentinvention also include variants in which a different base is present atone or more of the nucleotide positions in the compound. For example, ifthe first nucleotide is an adenine, variants may be produced whichcontain thymidine, guanosine, cytidine or other natural or unnaturalnucleotides at this position. This may be done at any of the positionsof the antisense compound. These compounds are then tested using themethods described herein to determine their ability to inhibitexpression of a target nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 50% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired. Such conditionsinclude, i.e., physiological conditions in the case of in vivo assays ortherapeutic treatment, and conditions in which assays are performed inthe case of in vitro assays.

An antisense compound, whether DNA, RNA, chimeric, substituted etc, isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarily to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the ease of in vivoassays or therapeutic treatment, and in the ease of in vitro assays,under conditions in which the assays are performed.

In another preferred embodiment, targeting of a Lipid transport andmetabolism gene including without limitation, antisense sequences whichare identified and expanded, using for example, PCR, hybridization etc.,one or more of the sequences set forth as SEQ ID NO: 8 to 22, and thelike, modulate the expression or function of a Lipid transport andmetabolism gene. In one embodiment, expression or function isup-regulated as compared to a control. In another preferred embodiment,expression or function is down-regulated as compared to a control.

In another preferred embodiment, oligonucleotides comprise nucleic acidsequences set forth as SEQ ID NOS: 23 to 263 including antisensesequences which are identified and expanded, using for example, PCR,hybridization etc. These oligonucleotides can comprise one or motemodified nucleotides, shorter or longer fragments, modified bonds andthe like. Examples of modified bonds or internucleotide linkagescomprise phosphorothioate, phosphorodithioate or the like. In anotherpreferred embodiment, the nucleotides comprise a phosphorus derivative.The phosphorus derivative (or modified phosphate group) which may beattached to the sugar or sugar analog moiety in the modifiedoligonucleotides of the present invention may be a monophosphate,diphosphate, triphosphate, alkylphosphate, alkanephosphate,phosphorothioate and the like. The preparation of the above-notedphosphate analogs, and their incorporation into nucleotides, modifiednucleotides and oligonucleotides, per se, is also known and need not bedescribed here.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

In embodiments of the present invention oligomeric antisense compounds,particularly oligonucleotides, bind to target nucleic acid molecules andmodulate the expression and/or function of molecules encoded by a targetgene. The functions of DNA to be interfered comprise, for example,replication and transcription. The functions of RNA to be interferedcomprise all vital functions such as, for example, translocation of theRNA to the site of protein translation, translation of protein from theRNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The functions may be up-regulated or inhibited depending on thefunctions desired.

The antisense compounds, include, antisense oligomeric compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, primers, probes, and otheroligomeric compounds that hybridize to at least a portion of the targetnucleic acid. As such, these compounds may be introduced in the form ofsingle-stranded, double-stranded, partially single-stranded, or circularoligomeric compounds.

Targeting an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes a Lipid transport andmetabolism gene.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

In a preferred embodiment, the antisense oligonucleotides bind to thenatural antisense sequences of a Lipid transport and metabolism gene andmodulate the expression and/or function of a Lipid transport andmetabolism gene (SEQ ID NO: 1). Examples of antisense sequences includeSEQ ID NOS: 8 to 263.

In another preferred embodiment, the antisense oligonucleotides bind toone or more segments of a Lipid transport and metabolism genepolynucleotide and modulate the expression and/or function of a Lipidtransport and metabolism gene. The segments comprise at least fiveconsecutive nucleotides of a Lipid transport and metabolism gene senseor antisense polynucleotides.

In another preferred embodiment, the antisense oligonucleotides arespecific for natural antisense sequences of a Lipid transport andmetabolism gene wherein binding of the oligonucleotides to the naturalantisense sequences of a Lipid transport and metabolism gene modulateexpression and/or function of a Lipid transport and metabolism gene.

In another preferred embodiment, oligonucleotide compounds comprisesequences set forth as SEQ ID NOS: 23 to 263, antisense sequences whichare identified and expanded, using for example, PCR, hybridization etc.These oligonucleotides can comprise one or more modified nucleotides,shorter or longer fragments, modified bonds and the like. Examples ofmodified bonds or internucleotide linkages comprise phosphorothioate,phosphorodithioate or the like. In another preferred embodiment, thenucleotides comprise a phosphorus derivative. The phosphorus derivative(or modified phosphate group) which may be attached to the sugar orsugar analog moiety in the modified oligonucleotides of the presentinvention may be a monophosphate, diphosphate, triphosphate,alkylphosphate, alkanephosphate, phosphorothioate and the like. Thepreparation of the above-noted phosphate analogs, and theirincorporation into nucleotides, modified nucleotides andoligonucleotides, per se, is also known and need not be described here.

Since, as is known in the art, the translation initiation codon istypically 5-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes has a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG; and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to junction in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or mote alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA transcribed from a geneencoding a Lipid transport and metabolism gene, regardless of thesequences) of such codons. A translation lamination codon (or “stopcodon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAGand 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions that may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, atargeted region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Another target region includes the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene). Still another target regionincludes the 3′ untranslated region (3′UTR), known in the art to referto the portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA (or correspondingnucleotides on the gene). The 5′ cap site of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap site. Another target region for thisinvention is the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. In one embodiment, targeting splicesites, i.e., intron-exon junctions or exoneration junctions, isparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. An aberrant fusion junction due to rearrangementor deletion is another embodiment of a target site. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. Introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

In another preferred embodiment, the antisense oligonucleotides bind tocoding and/or non-coding regions of a target polynucleotide and modulatethe expression and/or function of the target molecule.

In another preferred embodiment, the antisense oligonucleotides bind tonatural antisense polynucleotides and modulate the expression and/orfunction of the target molecule.

In another preferred embodiment, the antisense oligonucleotides bind tosense polynucleotides and modulate the expression and/or function of thetarget molecule.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame genomic DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, (hereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also embodiments of target nucleic acids.

The locations on the target nucleic acid to which the antisensecompounds hybridize are defined as at least a 5-nucleotide long portionof a target region to which an active antisense compound is targeted.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure.

Target segments 5-100 nucleotides in length comprising a stretch of atleast five (5) consecutive nucleotides selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 5 consecutive nucleotides from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleotides beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 5 to about 100 nucleotides). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 5 consecutive nucleotides from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleotides being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 5 to about 100nucleotides). One having skill in the art armed with the target segmentsillustrated herein will be able, without undue experimentation, toidentify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

In embodiments of the invention the oligonucleotides bind to anantisense strand of a particular target. The oligonucleotides are atleast 5 nucleotides in length and can be synthesized so eacholigonucleotide targets overlapping sequences such that oligonucleotidesare synthesized to cover the entire length of the target polynucleotide.The targets also include coding as well as non coding regions.

In one embodiment it is preferred to target specific nucleic acids byantisense oligonucleotides. Targeting an antisense compound to aparticular nucleic acid, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a non coding polynucleotidesuch as for example, non coding RNA (ncRNA).

RNAs can be classified into (1) messenger RNAs (mRNAs), which aretranslated into proteins, and (2) non-protein-coding RNAs (ncRNAs).ncRNAs comprise microRNAs, antisense transcripts and otherTranscriptional Units (TU) containing a high density of stop codons andlacking any extensive “Open Reading Frame”. Many neRNAs appear to startfrom initiation sites in 3′ untranslated regions (3′UTRs) ofprotein-coding loci. neRNAs are often rare and at least half of theneRNAs that have been sequenced by the FANTOM consortium seem not to bepolyadenylated. Most researchers have for obvious reasons focused onpolyadenylated mRNAs that are processed and exported to the cytoplasm.Recently, it was shown that the set of non-polyadenylated nuclear RNAsmay be very large, and that many such transcripts arise from so-calledintergenic regions (Cheng, J. et al. (2005) Science 308 (5725),1149-1154; Kapranov, P. et al. (2005). Genome Res 15 (7), 987-997). Themechanism by which neRNAs may regulate gene expression is by basepairing with target transcripts. The RNAs that function by base pairingcan be grouped into (1) cis encoded RNAs that are encoded at the samegenetic location, but on the opposite strand to the RNAs they act uponand therefore display perfect complementarity to their target, and (2)trans-encoded RNAs that are encoded at a chromosomal location distinctfrom the RNAs they act upon and generally do not exhibit perfectbase-pairing potential with their targets.

Without wishing to be bound by theory, perturbation of an antisensepolynucleotide by the antisense oligonucleotides described herein canalter the expression of the corresponding sense messenger RNAs. However,this regulation can either be discordant (antisense knockdown results inmessenger RNA elevation) or concordant (antisense knockdown results inconcomitant messenger RNA reduction). In these cases, antisenseoligonucleotides can be targeted to overlapping or non-overlapping partsof the antisense transcript resulting in its knockdown or sequestration.Coding as well as non-coding antisense can be targeted in an identicalmanner and that either category is capable of regulating thecorresponding sense transcripts-either in a concordant or disconcordantmanner. The strategies that are employed in identifying newoligonucleotides for use against a target can be based on tire knockdownof antisense RNA transcripts by antisense oligonucleotides or any othermeans of modulating the desired target.

Strategy 1: In the ease of discordant regulation, knocking down theantisense transcript elevates the expression of the conventional (sense)gene. Should that latter gene encode for a known or putative drugtarget, then knockdown of its antisense counterpart could conceivablymimic the action of a receptor agonist or an enzyme stimulant.

Strategy 2: in the case of concordant regulation, one couldconcomitantly knock down both antisense and sense transcripts andthereby achieve synergistic reduction of the conventional (sense) geneexpression. If, for example, an antisense oligonucleotide is used toachieve knockdown, then this strategy can be used to apply one antisenseoligonucleotide targeted to the sense transcript and another antisenseoligonucleotide to the corresponding antisense transcript, or a singleenergetically symmetric antisense oligonucleotide that simultaneouslytargets overlapping sense and antisense transcripts.

According to the present invention, antisense compounds includeantisense oligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, and otheroligomeric compounds which hybridize to at least a portion of the targetnucleic acid and modulate its function. As such, they may be DNA, RNA,DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one ormore of these. These compounds may be single-stranded, doublestranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges, mismatches or loops.Antisense compounds are routinely prepared linearly but can be joined orotherwise prepared to be circular and/or branched. Antisense compoundscan include constructs such as, for example, two strands hybridized toform a wholly or partially double-stranded compound or a single strandwith sufficient self-complementarity to allow for hybridization andformation of a fully or partially double-stranded compound. The twostrands can be linked internally leaving free 3′ or 5′ termini or can belinked to form a continuous hairpin structure or loop. The hairpinstructure may contain an overhang on either the 5′ or 3′ terminusproducing an extension of single stranded character. The double strandedcompounds optionally can include overhangs on the ends. Furthermodifications can include conjugate groups attached to one of thetermini, selected nucleotide positions, sugar positions or to one of theinternucleoside linkages. Alternatively, the two strands can be linkedvia a non-nucleic acid moiety or linker group. When formed from only onestrand, dsRNA can take the form of a self-complementary hairpin-typemolecule that doubles back on itself to form a duplex. Thus, the dsRNAscan be fully or partially double stranded. Specific modulation of geneexpression can be achieved by stable expression of dsRNA hairpins intransgenic cell lines, however, in some embodiments, the gene expressionor function is up regulated. When formed from two strands, or a singlestrand that takes the form of a self-complementary hairpin-type moleculedoubled back on itself to form a duplex, the two strands (orduplex-forming regions of a single strand) are complementary RNA strandsthat base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

In another preferred embodiment, the desired oligonucleotides orantisense compounds, comprise at least one of: antisense RNA, antisenseDNA, chimeric antisense oligonucleotides, antisense oligonucleotidescomprising modified linkages, interference RNA (RNAi), short interferingRNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA(stRNA); or a short, hairpin RNA (shRNA); small RNA-induced geneactivation (RNAa); small activating RNAs (saRNAs), or combinationsthereof.

dsRNA can also activate gene expression, a mechanism that has beentermed “small RNA-induced gene activation” or RNAa. dsRNAs targetinggene promoters induce potent transcriptional activation of associatedgenes. RNAa was demonstrated in human cells using synthetic dsRNAs,termed “small activating RNAs” (saRNAs). It is currently not knownwhether RNAa is conserved in other organisms.

Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA)and microRNA (miRNA), have been found to be the trigger of anevolutionary conserved mechanism known as RNA interference (RNAi). RNAiinvariably leads to gene silencing via remodeling chromatin to therebysuppress transcription, degrading complementary mRNA, or blockingprotein translation. However, in instances described in detail in theexamples section which follows, oligonucleotides are shown to increasethe expression and/or function of the Lipid transport and metabolismgene polynucleotides and encoded products thereof. dsRNAs may also actas small activating RNAs (saRNA). Without wishing to be bound by theory,by targeting sequences in gene promoters, saRNAs would induce targetgene expression in a phenomenon referred to as dsRNA-inducedtranscriptional activation (RNAa).

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of a Lipid transport and metabolism genepolynucleotide. “Modulators” are those compounds that decrease orincrease the expression of a nucleic acid molecule encoding a Lipidtransport and metabolism gene and which comprise at least a 5-nucleotideportion that is complementary to a preferred target segment. Thescreening method comprises the steps of contacting a preferred targetsegment of a nucleic acid molecule encoding sense or natural antisensepolynucleotides of a Lipid transport and metabolism gene with one ormore candidate modulators, and selecting for one or more candidatemodulators which decrease or increase the expression of a nucleic acidmolecule encoding a Lipid transport and metabolism gene polynucleotide,e.g. SEQ ID NOS: 23 to 263. Once it is shown that the candidatemodulator or modulators are capable of modulating (e.g. eitherdecreasing or increasing) the expression of a nucleic acid moleculeencoding a Lipid transport and metabolism gene polynucleotide, themodulator may then be employed in further investigative studies of thefunction of a Lipid transport and metabolism gene polynucleotide, or foruse as a research, diagnostic, or therapeutic agent in accordance withthe present invention.

Targeting the natural antisense sequence preferably modulates thefunction of the target gene. For example, the Lipid transport andmetabolism gene (e.g. accession numbers NM.sub.--005502,NM.sub.--000229, NM.sub.--002332, NM.sub.--008512, NM.sub.--000527.3,NM.sub.--000041, NM.sub.--000039, FIG. 2). In a preferred embodiment,the target is an antisense polynucleotide of the Lipid transport andmetabolism gene. In a preferred embodiment, an antisense oligonucleotidetargets sense and/or natural antisense sequences of a Lipid transportand metabolism gene polynucleotide (e.g. accession numbersNM.sub.--005502, NM.sub.--000229, NM.sub.--002332, NM.sub.--008512,NM.sub.--000527, NM.sub.--000041, NM.sub.--000039, FIG. 2), variants,alleles, isoforms, homologs, mutants, derivatives, fragments andcomplementary sequences thereto. Preferably the oligonucleotide is anantisense molecule and the targets include coding and noncoding regionsof antisense and/or sense Lipid transport and metabolism genepolynucleotides.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

(Such double stranded oligonucleotide moieties have been shown in theart to modulate target expression and regulate translation as well asRNA processing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., (1998)Nature, 391, 806-811; Timmons and Fire, (1998) Nature, 395, 854; Timmonset al., (2001) Gene, 263, 103-112; Tabara et al., (1998) Science, 282,430-431; Montgomery et al., (1998) Proc. Natl. Acad. Sci. USA, 95,15502-15507; Tuschl et al., (1999) Genes Dev., 13, 3191-3197; Elbashiret al., (2001) Nature, 411, 494-498; Elbashir et al., (2001) Genes Dev.15, 188-200). For example, such double-stranded moieties have been shownto inhibit the target by the classical hybridization of antisense strandof the duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., (2002) Science, 295, 694-697).

In a preferred embodiment, an antisense oligonucleotide targets Lipidtransport and metabolism gene polynucleotides (e.g. accession numbersNM.sub.--005502, NM.sub.--000229, NM.sub.--002332, NM.sub.--008512.NM.sub.--000527, NM.sub.--000041, NM.sub.--000039), variants, alleles,isoforms, homologs, mutants, derivatives, fragments and complementarysequences thereto. Preferably the oligonucleotide is an antisensemolecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to Lipid transport and metabolism gene alone butextends to any of the isoforms, receptors, homologs and the like of aLipid transport and metabolism gene molecule.

In another preferred embodiment, an oligonucleotide targets a naturalantisense sequence of a Lipid transport and metabolism genepolynucleotide, for example, polynucleotides set forth as SEQ ID NO: 8to 22, and any variants, alleles, homologs, mutants, derivatives,fragments and complementary sequences thereto. Examples of antisenseoligonucleotides are set forth as SEQ ID NOS: 23 to 263.

In one embodiment, the oligonucleotides are complementary to or bind tonucleic acid sequences of a Lipid transport and metabolism geneantisense, including without limitation noncoding sense and/or antisensesequences associated with a Lipid transport and metabolism genepolynucleotide and modulate expression and/or function of a Lipidtransport and metabolism gene molecule.

In another preferred embodiment, the oligonucleotides are complementaryto or bind to nucleic acid sequences of a Lipid transport and metabolismgene natural antisense, set forth as SEQ ID NO: 8 to 22 and modulateexpression and/or function of a Lipid transport and metabolism genemolecule.

In a preferred embodiment, oligonucleotides comprise sequences of atleast 5 consecutive nucleotides of SEQ ID NOS: 23 to 263 and modulateexpression and/or function of a Lipid transport and metabolism genemolecule.

The polynucleotide targets comprise Lipid transport and metabolism gene,including family members thereof, variants of a Lipid transport andmetabolism gene; mutants of a Lipid transport and metabolism gene,including SNPs; noncoding sequences of a Lipid transport and metabolismgene; alleles of a Lipid transport and metabolism gene; speciesvariants, fragments and the like. Preferably the oligonucleotide is anantisense molecule.

In another preferred embodiment, the oligonucleotide targeting Lipidtransport and metabolism gene polynucleotides, comprise: antisense RNA,interference RNA (RNAi), short interfering RNA (siRNA); microinterfering RNA (miRNA); a small, temporal RNA (stRNA); or a short,hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); or, smallactivating RNA (saRNA).

In another preferred embodiment, targeting of a Lipid transport andmetabolism gene polynucleotide, e.g. SEQ ID NO: 8 to 22 modulate theexpression or function of these targets. In one embodiment, expressionor function is up-regulated as compared to a control. In anotherpreferred embodiment, expression or function is down-regulated ascompared to a control.

In another preferred embodiment, antisense compounds comprise sequencesset forth as SEQ ID NOS: 23 to 263. These oligonucleotides can compriseone or more modified nucleotides, shorter or longer fragments, modifiedbonds and the like.

In another preferred embodiment, SEQ ID NOS: 23 to 263 comprise one ormore LNA nucleotides.

The modulation of a desired target nucleic acid can be carried out inseveral ways known in the art. For example, antisense oligonucleotides,siRNA etc. Enzymatic nucleic acid molecules (e.g., ribozymes) arenucleic acid molecules capable of catalyzing one or more of a variety ofreactions, including the ability to repeatedly cleave other separatenucleic acid molecules in a nucleotide base sequence-specific manner.Such enzymatic nucleic acid molecules can be used, for example, totarget virtually any RNA transcript (Zaug et al., 324, Nature 429 15186;Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic AcidsResearch 1371, 1989).

Because of their sequence-specificity, trans-cleaving enzymatic nucleicacid molecules show promise as therapeutic agents for human disease(Usman & McSwiggcn, (1995) Ann. Rep. Med. Chem. 30, 285-294;Christofiersen and Marr, (1995) J. Med. Chem. 38, 2023-2037). Enzymaticnucleic acid molecules can be designed to cleave specific RNA targetswithin the background of cellular RNA. Such a cleavage event renders themRNA non-functional and abrogates protein expression from that RNA. Inthis manner, synthesis of a protein associated with a disease state canbe selectively inhibited.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution) strategies(Orgel, (1979) Proc. R. Soc. London, B 205, 435) haw been used to evolvenew nucleic acid catalysts capable of catalyzing a variety of reactions,such as cleavage and ligation of phosphodiester linkages and amidelinkages, (Joyce, (1989) Gene, 82, 83-87; Beaudry et al., (1992) Science257, 635-641; Joyce, (1992) Scientific American 267, 90-97; Breaker etal., (1994) TIBTECH 12, 268; Bartel et al, (1993) Science 261:1411-1418;Szostak, (1993) TIBS 17, 89-93; Kumar et al, (1995) FASEB J., 9, 1183;Breaker, (1996) Curr. Op. Biotech., 7, 442).

The development of ribozymes that are optimal for catalytic activitywould contribute significantly to any strategy that employs RNA-cleavingribozymes for the purpose of regulating gene expression. The hammerheadribozyme, for example, functions with a catalytic rate (kcat) of about 1min-1 in the presence of saturating (10 mM) concentrations of Mg2+cofactor. An artificial “RNA ligase” ribozyme has been shown to catalyzethe corresponding self-modification reaction with a rate of about 100min-1. In addition, it is known that certain modified hammerheadribozymes that have substrate binding arms made of DNA catalyze RNAcleavage with multiple turn-over rates that approach 100 min−1. Finally,replacement of a specific residue within the catalytic core of thehammerhead with certain nucleotide analogues gives modified ribozymesthat show as much as a 10-fold improvement in catalytic rate. Thesefindings demonstrate that ribozymes can promote chemical transformationswith catalytic rates that are significantly greater than those displayedin vitro by most natural selfcleaving ribozymes. It is then possiblethat the structures of certain selfcleaving ribozymes may be optimizedto give maximal catalytic activity, or that entirely new RNA motifs canbe made that display significantly faster rates for RNA phosphodiestercleavage.

Intermolecular cleavage of an RNA substrate by an RNA catalyst that fitsthe “hammerhead” model was first shown in 1987 (Uhlenbeck, O. C. (1987)Nature, 328: 596-600). The RNA catalyst was recovered and reacted withmultiple RNA molecules, demonstrating that it was truly catalytic.

Catalytic RNAs designed based on the “hammerhead” motif have been usedto cleave specific target sequences by making appropriate base changesin the catalytic RNA to maintain necessary base pairing with the targetsequences (Haseloff and Gerlach, (1988) Nature, 334, 585; Walbot andBruening, (1988) Nature, 334, 196; Uhlenbeck, O. C. (1987) Nature, 328:596-600; Koizumi, M., et al. (1988) FEBS Lett., 228: 228-230). This hasallowed use of the catalytic RNA to cleave specific target sequences andindicates that catalytic RNAs designed according to the “hammerhead”model may possibly cleave specific substrate RNAs in vivo, (sec Haseloffand Gerlach, (1988) Nature, 334, 585; Walbot and Bruening, (1988)Nature, 334, 196; Uhlenbeck, O. C. (1987) Nature, 328: 596-600).

RNA interference (RNAi) has become a powerful tool for modulating geneexpression in mammals and mammalian cells. This approach requires thedelivery of small interfering RNA (siRNA) either as RNA itself or asDNA, using an expression plasmid or virus and the coding sequence forsmall hairpin RNAs that are processed to siRNAs. This system enablesefficient transport of the pre-siRNAs to the cytoplasm where they areactive and permit the use of regulated and tissue specific promoters forgene expression.

In a preferred embodiment, an oligonucleotide or antisense compoundcomprises an oligomer or polymer of ribonucleic acid (RNA) and/ordeoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homologthereof. This term includes oligonucleotides composed of naturallyoccurring nucleotides, sugars and covalent internucleoside (backbone)linkages as well as oligonucleotides having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often desired over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for a target nucleic acid and increased stability inthe presence of nucleases.

According to the present invention, the oligonucleotides or “antisensecompounds” include antisense oligonucleotides (e.g. RNA, DNA, mimetic,chimera, analog or homolog thereof), ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, saRNA, aRNA, andother oligomeric compounds which hybridize to at least a portion of thetarget nucleic acid and modulate its function. As such, they may be DNA,RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of oneor more of these. These compounds may be single-stranded,double-stranded, circular or hairpin oligomeric compounds and maycontain structural elements such as internal or terminal bulges,mismatches or loops. Antisense compounds are routinely prepared linearlybut can be joined or otherwise prepared to be circular and/or branched.Antisense compounds can include constructs such as, fix example, twostrands hybridized to form a wholly or partially double-strandedcompound or a single strand with sufficient self-complementarity toallow for hybridization and formation of a fully or partiallydouble-stranded compound. The two strands can be linked internallyleaving free 3′ or 5′ termini or can be linked to form a continuoushairpin structure or loop. The hairpin structure may contain an overhangon either the 5′ or 3′ terminus producing an extension of singlestranded character. The double stranded compounds optionally can includeoverhangs on the ends. Further modifications can include conjugategroups attached to one of the termini, selected nucleotide positions,sugar positions or to one of the internucleoside linkages.Alternatively, the two strands can be linked via a non-nucleic acidmoiety or linker group. When formed from only one strand, dsRNA can takethe form of a self-complementary hairpin-type molecule that doubles backon itself to form a duplex. Thus, the dsRNAs can be fully or partiallydouble stranded. Specific modulation of gene expression can be achievedby stable expression of dsRNA hairpins in transgenic cell lines (Hammondet al., (1991) Nat. Rev. Genet., 2, 110-119; Matzke et al., (2001) Curr.Opin. Genet. Dev., 11, 221-227; Sharp, (2001) Genes Dev., 15, 485-490).When formed from two strands, or a single strand that takes the form ofa self-complementary hairpin-type molecule doubled back on itself toform a duplex, the two strands (or duplex-forming regions of a singlestrand) are complementary RNA strands that base pair in Watson-Crickfashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

The antisense compounds in accordance with this invention can comprisean antisense portion from about 5 to about 80 nucleotides (i.e. fromabout 5 to about 80 linked nucleosides) in length. This refers to thelength of the antisense strand or portion of the antisense compound. Inother words, a single-stranded antisense compound of the inventioncomprises from 5 to about 80 nucleotides, and a double-strandedantisense compound of the invention (such as a dsRNA, for example)comprises a sense and an antisense strand or portion of 5 to about 80nucleotides in length. One of ordinary skill in the art will appreciatethat this comprehends antisense portions of 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides inlength, or any range therewithin.

In one embodiment, the antisense compounds of the invention haveantisense portions of 10 to 50 nucleotides in length. One havingordinary skill in the art will appreciate that this embodiesoligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31.32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length, or any range therewithin. In some embodiments,the oligonucleotides are 15 nucleotides in length.

In one embodiment, the antisense or oligonucleotide compounds of theinvention have antisense portions of 12 or 13 to 30 nucleotides inlength. One having ordinary skill in the art will appreciate that thisembodies antisense compounds having antisense portions of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length, or any range therewithin.

In another preferred embodiment, the oligomeric compounds of the presentinvention also include variants in which a different base is present atone or more of the nucleotide positions in the compound. For example, ifthe first nucleotide is an adenosine, variants may be produced whichcontain thymidine, guanosine or cytidine at this position. This may bedone at any of the positions of the antisense or dsRNA compounds. Thesecompounds are then tested using the methods described herein todetermine their ability to inhibit expression of a target nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 40% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

In another preferred embodiment, the antisense oligonucleotides, such asfor example, nucleic acid molecules set forth in SEQ ID NOS: 8 to 263comprise one or more substitutions or modifications, in one embodiment,the nucleotides are substituted with locked nucleic acids (LNA).

In another preferred embodiment, the oligonucleotides target one or moreregions of the nucleic acid molecules sense and/or antisense of codingand/or non-coding sequences associated with Lipid transport andmetabolism gene and the sequences set forth as SEQ ID NOS: 1 to 7 and 8to 22. The oligonucleotides are also targeted to overlapping regions ofSEQ ID NOS: 1 to 7 and 8 to 22.

Certain preferred oligonucleotides of this invention are chimericoligonucleotides. “Chimeric oligonucleotides” or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the target) and a region thatis a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of antisense modulation of gene expression.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligonucleotides are used, compared tophosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the an. In one preferred embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in thiscase, a nucleic acid encoding ras) is routinely determined by measuringthe Tm of an oligonucleotide/target pair, which is the temperature atwhich the oligonucleotide and target dissociate; dissociation isdetected spectrophotometrically. The higher the Tm, the greater is theaffinity of the oligonucleotide for the target.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotides mimetics as described above.Such; compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In another preferred embodiment, the region of the oligonucleotide whichis modified comprises at least one nucleotide modified at the 2′position of the sugar, most preferably a 2′-Oalkyl, 2′-O-alkyl-O-alkylor 2′-fluoro-modified nucleotide. In other preferred embodiments, RNAmodifications include 2-fluoro, 2′-amino and 2′ O-methyl modificationson the ribose of pyrimidines, abasic residues or an inverted base at the3′ end of the RNA. Such modifications are routinely incorporated intooligonucleotides and these oligonucleotides have been shown to have ahigher Tm (i.e., higher target binding affinity) than;2′-deoxyoligonucleotides against a given target. The effect of suchincreased affinity is to greatly enhance RNAi oligonucleotide inhibitionof gene expression. RNAse H is a cellular endonuclease that cleaves theRNA strand of RNA:DNA duplexes; activation of this enzyme thereforeresults in cleavage of the RNA target, and thus can greatly enhance theefficiency of RNAi inhibition. Cleavage of the RNA target can beroutinely demonstrated by gel electrophoresis. In another preferredembodiment, the chimeric oligonucleotide is also modified to enhancenuclease resistance. Cells contain a variety of exo- and endo-nucleaseswhich can degrade nucleic acids. A number of nucleotide and nucleosidemodifications have been shown to make the oligonucleotide into whichthey are incorporated more resistant to nuclease digestion than thenative oligodeoxynucleotide. Nuclease resistance is routinely measuredby incubating oligonucleotides with cellular extracts or isolatednuclease solutions and measuring the extent of intact oligonucleotideremaining over time, usually by gel electrophoresis. Oligonucleotideswhich have been modified to enhance their nuclease resistance surviveintact for a longer time than unmodified oligonucleotides. A variety ofoligonucleotide modifications have been demonstrated to enhance orconfer nuclease resistance. Oligonucleotides which contain at least onephosphorothioate modification are presently more preferred. In somecases, oligonucleotide modifications which enhance target bindingaffinity are also, independently, able to enhance nuclease resistance.Some desirable modifications can be found in De Mesmaeker et al. (1995)Acc. Chem. Res., 28:366-374.

Specific examples of some preferred oligonucleotides envisioned for thisinvention include those comprising modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioate backbones and those with heteroatom backbones,particularly CH2-NH—O—CH2, CH, —N(CH3)-O—CH2 (known as amethylene(methylimino) or MMI backbone), CH2-O—N (CH3-CH2,CH2-N(CH3)-N(CH3)-CH2 and O—N(CH3)-CH2-CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH). The amide backbonesdisclosed by Dc Mesmaeker et al. (1995) Acc. Chem. Res. 28:366-374 arealso preferred. Also preferred are oligonucleotides having morpholinobackbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506). Inother preferred embodiments, such as the peptide nucleic acid (PNA)backbone, the phosphodiester backbone of the oligonucleotide is replacedwith a polyamide backbone, the nucleotides being bound directly orindirectly to the aza nitrogen atoms of the polyamide backbone (Nielsenet al. (1991) Science 254, 1497). Oligonucleotides may also comprise oneor more substituted sugar moieties. Preferred oligonucleotides compriseone of the following at the 2′ position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3 where n is from 1 toabout 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O—, S, or N-alkyl; O—, S, orN-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2′-methoxyethoxy [2′-O—CH2 CH2 OCH3, also known as2′-O-(2-methoxyethyl)] (Martin et al., (1995) Helv. Chim. Acta, 78,486). Other preferred modifications include 2′-methoxy (2′-O—CH3),2′-propoxy (2-OCH2 CH2CH3) and 2′-fluoro (2-F). Similar modificationsmay also be made at other positions on the oligonucleotide, particularlythe 3′ position of the sugar on the 3′ terminal nucleotide and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyls in place of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively,nucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleotidesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleotides include nucleotides found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 3-Me-C), 5-hydroxymethylcytosine (BMC), glycosyl HMC andgentobiosyl BMC, as well as synthetic nucleotides, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalkylamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and2,6-diaminopurine. (Kornberg, A., DNA Replication, W. H. Freeman & Co.,San Francisco, 1980, pp 75-77; Gebeyehu, G., (1987) et al. Nucl. AcidsRes. 15:4513). A “universal” base known in the art, e.g., inosine, maybe included. 5-Me-C substitutions have been shown to increase nucleicacid duplex stability by 0.6-1,2.degree. C. (Sanghvi, Y. S., in Crooke,S. T, and Lebleu, B., eds., Antisense Research and Applications, CRCPress, Boca Raton, 1993, pp. 276-278) and are presently preferred basesubstitutions.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, a cholesteryl moiety (Letsingeret al., (1989) Proc. Natl. Acad. Sci. USA 86, 6553), cholic acid(Manoharan et al. (1994) Bioorg. Med. Chem. Let. 4, 1053), a thioether,e.g., hexyl-S-tritylthiol (Manoharan et al. (1992) Ann. N.Y. Acad. Sci.660, 306; Manoharan et al. (1993) Bioorg. Med. Chem. Let 3, 2765), athiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res. 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al. EMBO J. 1991, 10, 111; Kabanov et al. (1990) FEBS Lett. 259, 327;Svinarchuk et al. (1993) Biochimie 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glyccrol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al. (1995)Tetrahedron Lett. 36, 3651; Shea et al. (1990) Nucl. Acids Res. 18,3777), a polyamine or a polyethylene glycol chain (Manoharan et al.(1995) Nucleosides & Nucleotides, 14, 969), or adamantane acetic acid(Manoharan et al. (1995) Tetrahedron Lett 36, 3651). Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides are known in the art, for example, U.S. Pat. Nos.5,138,045, 5,218,105 and 5,459,255.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes oligonucleotides which are chimericoligonucleotides as hereinbefore defined.

In another embodiment, the nucleic acid molecule of the presentinvention is conjugated with another moiety including but not limited toabasic nucleotides, polyether, polyamine, polyamides, peptides,carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in theart will recognize that these molecules can be linked to one or more ofany nucleotides comprising the nucleic acid molecule at severalpositions on the sugar, base or phosphate group.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of one of ordinary drill in the art. It is alsowell known to use similar techniques to prepare other oligonucleotidessuch as the phosphorothioates and alkylated derivatives. It is also wellknown to use similar techniques and commercially available modifiedamidites and controlled-pore glass (CPG) products such as biotin,fluorescein, acridine or psoralen-modified amidites and/or CPG(available from Glen Research, Sterling Va.) to synthesize fluorescentlylabeled, biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

In accordance with the invention, use of modifications such as the useof LNA monomers to enhance the potency, specificity and duration ofaction and broaden the routes of administration of oligonucleotidescomprised of current chemistries such as MOE, ANA, FANA, PS etc (Uhlman,et al. (2000) Current Opinions in Drug Discovery & Development Vol. 3 No2). This can be achieved by substituting some of the monomers in thecurrent oligonucleotides by LNA monomers. The LNA modifiedoligonucleotide may have a size similar to the parent compound or may belarger or preferably smaller. It is preferred that such LNA-modifiedoligonucleotides contain less than about 70%, more preferably less thanabout 60%, most preferably less than about 50% LNA monomers and thattheir sizes are between about 5 and 25 nucleotides, more preferablybetween about 12 and 20 nucleotides.

Preferred modified oligonucleotide backbones comprise, but not limitedto, phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3-5′ to 5′-3′ or 2′-5‘ to S’-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus containing linkages comprise, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholine linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides comprise, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds comprise, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen, et al. (1991) Science 254, 1497-1500.

In another preferred embodiment of the invention the oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH2-NH—O—CH2-, —CH2-N(CH3)-O—CH2-known asa methylene (methylimino) or MMI backbone, —CH2-O—N(CH3)-CH2-,—CH2N(CH3)-N(CH3) CH2- and —O—N(CH3)-CH2-CH2- wherein the nativephosphodiester backbone is represented as —O—P—O—CH2- of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C to (TO alkyl or C2 to CO alkenyland alkynyl. Particularly preferred are O (CH2)n OmCH3, O(CH2)n, OCH3,O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2nON(CH2)nCH3)2 where n andm can be from 1 to about 10. Other preferred oligonucleotides compriseone of the following at the 2′ position: C to CO, (lower alkyl,substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification comprises 2′-methoxyethoxy (2′-O—CH2CH2OCH3,also known as 2′-O-(2-methoxyethyl) or 2-MOE) (Martin et al., (1995)Helv. Chim. Acta, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification comprises 2-dimethylaminooxyethoxy, i.e., aO(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examplesherein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2.

Other preferred modifications comprise 2′-methoxy (2′-OCH3),2′-aminopropoxy (2-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures comprise, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646, 265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference.

Oligonucleotides may also comprise nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleotides comprise the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleotides comprise other synthetic andnatural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleotides comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., ‘AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, ‘Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleotides are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Thesecomprise Substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2.degree. C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds. ‘Antisense Researchand Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-Omethoxyethyl sugar modifications.

Representative United States patents that teach the preparation of theabove noted modified nucleotides as well as other modified nucleotidescomprise, but are not limited to, U.S. Pat. No. 3,687,808, as well asU.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941,each of which is herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates, which enhance the activity, cellular distribution, orcellular uptake of the oligonucleotide.

Such moieties comprise but are not limited to, lipid moieties such as acholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA,86, 6553-6556), cholic acid (Manoharan et al., (1994) Bioorg. Med. Chem.Let., 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., (1992) Ann. N. Y. Acad. Sci., 660, 306-309; Manoharan et al.,(1993) Bioorg. Med. Chem. Let, 3, 2765-2770), a thiocholesterol(Oberhauser et al., (1992) Nucl. Acids Res., 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Kabanov et al., (1990)FEBS Lett, 259, 327-330; Svinarchuk et al., (1993) Biochimie 75, 49-54),a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., (1995)Tetrahedron Lett, 36, 3651-3654; Shea et al., (1990) Nucl. Acids Res.,18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan etal., (1995) Nucleosides & Nucleotides, 14, 969-973), or adamantaneacetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36, 3651-3654),a palmityl moiety (Mishra et al., (1995) Biochim Biophys. Acta, 1264,229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterolmoiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277, 923-937).

Representative United States patents that teach the preparation of sucholigonucleotides conjugates comprise, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Drug Discovery:

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsdial exist between a Lipid transport and metabolism gene polynucleotideand a disease state, phenotype, or condition. These methods includedetecting or modulating a Lipid transport and metabolism genepolynucleotide comprising contacting a sample, tissue, cell, or organismwith the compounds of the present invention, measuring the nucleic acidor protein level of a Lipid transport and metabolism gene polynucleotideand/or a related phenotypic or chemical endpoint at some time aftertreatment, and optionally comparing the measured value to a non-treatedsample or sample treated with a further compound of the invention. Thesemethods can also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

Assessing Up-Regulation or Inhibition of Gene Expression:

Transfer of an exogenous nucleic acid into a host cell or organism canbe assessed by directly detecting the presence of the nucleic acid inthe cell or organism. Such detection can be achieved by several methodswell known in the art. For example, the presence of the exogenousnucleic acid can be detected by Southern blot or by a polymerase chainreaction (PCR) technique using primers that specifically amplifynucleotide sequences associated with the nucleic acid. Expression of theexogenous nucleic acids can also be measured using conventional methodsincluding gene expression analysis. For instance, mRNA produced from anexogenous nucleic acid can be detected and quantified using a Northernblot and reverse transcription PCR (RT-PCR).

Expression of RNA from the exogenous nucleic acid can also be detectedby measuring an enzymatic activity or a reporter protein activity. Forexample, antisense modulatory activity can be measured indirectly as adecrease or increase in target nucleic acid expression as an indicationthat the exogenous nucleic acid is producing the effector RNA. Based onsequence conservation, primers can be designed and used to amplifycoding regions of the target genes. Initially, the most highly expressedcoding region from each gene can be used to build a model control gene,although any coding or non coding region can be used. Each control geneis assembled by inserting each coding region between a reporter codingregion and its poly(A) signal. These plasmids would produce an mRNA witha reporter gene in the upstream portion of the gene raid a potentialRNAi target in the 3′ non-coding region. The effectiveness of individualantisense oligonucleotides would be assayed by modulation of thereporter gene. Reporter genes useful in the methods of the presentinvention include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP),cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracycline. Methods to determine modulation of areporter gene are well known in the art, and include, but are notlimited to, fluorometric methods (e.g. fluorescence spectroscopy,Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy),antibiotic resistance determination.

Lipid transport and metabolism gene protein and mRNA expression can beassayed using methods known to those of skill in the art and describedelsewhere herein. For example, immunoassays such as the ELISA can beused to measure protein levels. Lipid transport and metabolism geneantibodies for ELiSAs are available commercially, e.g., from R&D Systems(Minneapolis, Minn.), Abeam, Cambridge, Mass.

In embodiments, Lipid transport and metabolism gene expression (e.g.,mRNA or protein) in a sample (e.g., cells or tissues in vivo or invitro) treated using an antisense oligonucleotide of the invention isevaluated by comparison with Lipid transport and metabolism geneexpression in a control sample. For example, expression of the proteinor nucleic acid can be compared using methods known to those of skill inthe art with that in a mock-treated or untreated sample. Alternatively,comparison with a sample treated with a control antisenseoligonucleotide (e.g., one having an altered or different sequence) canbe made depending on the information desired. In another embodiment, adifference in the expression of the Lipid transport and metabolism geneprotein or nucleic acid in a treated vs. an untreated sample can becompared with the difference in expression of a different nucleic acid(including any standard deemed appropriate by the researcher, e.g., ahousekeeping gene) in a treated sample vs. an untreated sample.

Observed differences can be expressed as desired, e.g., in the form of aratio or fraction, for use in a comparison with control. In embodiments,the level of a Lipid transport and metabolism gene mRNA or protein, in asample treated with an antisense oligonucleotide of the presentinvention, is increased or decreased by about 1.25-fold to about 10-foldor more relative to an untreated sample or a sample treated with acontrol nucleic acid. In embodiments, the level of a Lipid transport andmetabolism gene mRNA or protein is increased or decreased by at leastabout 1.25-fold, at least about 1.3-fold, at least about 1.4-fold, atleast about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold,at least about 1.8-fold, at least about 2-fold, at least about 2.5-fold,at least about 3-fold, at least about 3.5-fold, at least about 4-fold,at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold,at least about 6-fold, at least about 6.5-fold, at least about 7-fold,at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold,at least about 9-fold, at least about 9.5-fold, or at least about10-fold or more.

Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics, and prophylaxis, and as research reagents and componentsof kits. Furthermore, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes orto distinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics and in various biological systems, thecompounds of the present invention, either alone or in combination withother compounds or therapeutics, are useful as tools in differentialand/or combinatorial analyses to elucidate expression patterns of aportion or the entire complement of genes expressed within cells andtissues.

As used herein the term “biological system” or “system” is defined asany organism, cell, cell culture or tissue that expresses, or is madecompetent to express products of the Lipid transport and metabolismgenes. These include, but are not limited to, humans, transgenicanimals, cells, cell cultures, tissues, xenografts, transplants andcombinations thereof.

As one non limiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, (2000) FEBS Lett., 480,17-24; Celis, et al., (2000) FEBS Lett, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., (2000) Drug Discov. Today,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, (1999) Methods Enzymol., 303, 258-72), TOGA(total gene expression analysis) (Sutcliffe, et al., (2000) Proc. Natl.Acad. Sci. U.S.A., 97, 1976-81), protein arrays and proteomics (Celis,et al., (2000) FEBS Lett., 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett, 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-37), subtractive RNA fingerprinting (SuRF) (Fuchs, et al.,(2000) Anal. Biochem. 286, 91-98; Larson, et al., (2000) Cytometry 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, (2000) Curr Opin. Microbiol. 3, 316-21), comparative genomichybridization (Carulli, et al., (1998) J. Cell Biochem. Suppl., 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, (1999) Eur. J. Cancer, 35, 1895-904) and mass spectrometrymethods (To, Comb. (2000) Chem. High Throughput Screen, 3, 235-41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding a Lipidtransport and metabolism gene. For example, oligonucleotides thathybridize with such efficiency and under such conditions as disclosedherein as to be effective Lipid transport and metabolism gene modulatorsare effective primers or probes under conditions favoring geneamplification or detection, respectively. These primers and probes areuseful in methods requiring the specific detection of nucleic acidmolecules encoding a Lipid transport and metabolism gene and in theamplification of said nucleic acid molecules for detection or for use infurther studies of a Lipid transport and metabolism gene. Hybridizationof the antisense oligonucleotides, particularly the primers and probes,of the invention with a nucleic acid encoding a Lipid transport andmetabolism gene can be detected by means known in the art. Such meansmay include conjugation of an enzyme to the oligonucleotide,radiolabeling of the oligonucleotide, or any other suitable detectionmeans. Kits using such detection means for detecting the level of aLipid transport and metabolism gene in a sample may also be prepared.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs have beensafely and effectively administered to humans and numerous clinicaltrials are presently underway. It is thus established that antisensecompounds can be useful therapeutic modalities that can be configured tobe useful in treatment regimes for the treatment of cells, tissues andanimals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofa Lipid transport and metabolism gene polynucleotide is treated byadministering antisense compounds in accordance with this invention. Forexample, in one non-limiting embodiment, the methods comprise the stepof administering to the animal in need of treatment, a therapeuticallyeffective amount of a Lipid transport and metabolism gene modulator. TheLipid transport and metabolism gene modulators of the present inventioneffectively modulate the activity of a Lipid transport and metabolismgene or modulate the expression of a Lipid transport and metabolism geneprotein. In one embodiment, the activity or expression of a Lipidtransport and metabolism gene in an animal is inhibited by about 10% ascompared to a control. Preferably, the activity or expression of a Lipidtransport and metabolism gene in an animal is inhibited by about 30%.More preferably, the activity or expression of a Lipid transport andmetabolism gene in an animal is inhibited by 50% or more. Thus, theoligomeric compounds modulate expression of a Lipid transport andmetabolism gene mRNA by at least 10%, by at least 50%, by at least 25%,by at least 30%, by at least 40%, by at least 50%, by at least 60%, byat least 70%, by at least 75%, by at least 80%, by at least 85%, by atleast 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%as compared to a control.

In one embodiment, the activity or expression of a Lipid transport andmetabolism gene and/or in an animal is increased by about 10% ascompared to a control. Preferably, the activity or expression of a Lipidtransport and metabolism gene in an animal is increased by about 30%.More preferably, the activity or expression of a Lipid transport andmetabolism gene in an animal is increased by 50% or mare. Thus, theoligomeric compounds modulate expression of a Lipid transport andmetabolism gene mRNA by at least 10%, by at least 50%, by at least 25%,by at least 30%, by at least 40%, by at least 50%, by at least 60%, byat least 70%, by at least 75%, by at least 80%, by at least 85%, by atleast 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%as compared to a control.

For example, the reduction of the expression of a Lipid transport andmetabolism gene may be measured in serum, blood, adipose tissue, liveror any other body fluid, tissue or organ of the animal. Preferably, thecells contained within said fluids, tissues or organs being analyzedcontain a nucleic acid molecule encoding Lipid transport and metabolismgene peptides and/or the Lipid transport and metabolism gene proteinitself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860, which are incorporated herein byreference. Conjugate moieties include, but are not limited to, lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic add, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

Although, the antisense oligonucleotides do not need to be administeredin the context of a vector in order to modulate a target expressionand/or function, embodiments of the invention relates to expressionvector constructs for the expression of antisense oligonucleotides,comprising promoters, hybrid promoter gene sequences and possess astrong constitutive promoter activity, or a promoter activity which canbe induced in the desired case.

In an embodiment, invention practice involves administering at least oneof the foregoing antisense oligonucleotides with a suitable nucleic aciddelivery system. In one embodiment, that system includes a non-viralvector operably linked to the polynucleotide. Examples of such nonviralvectors include the oligonucleotide alone (e.g. any one or more of SEQID NOS: 23 to 263) or in combination with a suitable protein,polysaccharide or lipid formulation.

Additionally suitable nucleic acid delivery systems include viralvector, typically sequence from at least one of an adenovirus,adenovirus-associated virus (AAV), helper-dependent adenovirus,retrovirus, or hemagglutination virus of Japan-liposome (HVJ) complex.Preferably, the viral vector comprises a strong eukaryotic promoteroperably linked to the polynucleotide e.g., a cytomegalovirus (CMV)promoter.

Additionally preferred vectors include viral vectors, fusion proteinsand chemical conjugates. Retroviral vectors include Moloney murineleukemia viruses and HIV-based viruses. One preferred HIV-based viralvector comprises at least two vectors wherein the gag and pol genes arefrom an HIV genome and the env gene is from another virus. DNA viralvectors are preferred. These vectors include pox vectors such asorthqpox or avipox vectors, herpesvirus vectors such as a herpes simplexI virus (HSV) vector [Geller, A. I. et al., (1995) J. Neurochem, 64:487; Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed.(Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al.,(1993) Proc Natl. Acad. Sci.: USA.: 90 7603; Geller, A. I., et al.,(1990) Proc Natl. Acad. Sci USA: 87:1149), Adenovirus Vectors (LeGalLaSalle et al., Science, 259:988 (1993); Davidson, et al., (1993) NatGenet. 3: 219; Yang, et al., (1995) J. Virol. 69: 2004) andAdeno-associated Virus Vectors (Kaplitt, M. G., et al., (1994) NatGenet. 8:148).

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are Anther described in U.S. Pat. No. 6,287,860,which is incorporated herein by reference.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

For treating tissues in the central nervous system, administration canbe made by, e.g., injection or infusion into the cerebrospinal fluid.Administration of antisense RNA into cerebrospinal fluid is described,e.g., in U.S. Pat. App. Pub. No. 2007/0117772, “Methods for slowingfamilial ALS disease progression,” incorporated herein by reference inits entirety.

When it is intended that the antisense oligonucleotide of the presentinvention be administered to cells in the central nervous system,administration can be with one or more agents capable of promotingpenetration of the subject antisense oligonucleotide across theblood-brain barrier. Injection can be made, e.g., in the entorhinalcortex or hippocampus. Delivery of neurotrophic factors byadministration of an adenovirus vector to motor neurons in muscle tissueis described in, e.g., U.S. Pat. No. 6,632,427,“Adenoviral-vector-mediated gene transfer into medullary motor neurons,”incorporated herein by reference. Delivery of vectors directly to thebrain, e.g., the striatum, the thalamus, the hippocampus, or thesubstantia nigra, is known in the art and described, e.g., in U.S. Pat.No. 6,756,523, “Adenovirus vectors for the transfer of foreign genesinto cells of the central nervous system particularly in brain,”incorporated herein by reference. Administration can be rapid as byinjection or made over a period of time as by slow infusion oradministration of slow release formulations.

The subject antisense oligonucleotides can also be linked or conjugatedwith agents that provide desirable pharmaceutical or pharmacodynamicproperties. For example, the antisense oligonucleotide can be coupled toany substance, known in the art to promote penetration or transportacross the blood-brain barrier, such as an antibody to the transferrinreceptor, and administered by intravenous injection. The antisensecompound can be linked with a vital vector, for example, that makes theantisense compound more effective and/or increases the transport of theantisense compound across the blood-brain barrier. Osmotic blood brainbarrier disruption can also be accomplished by, e.g., infusion of sugarsincluding, but not limited to, meso erythritol, xylitol, D(+) galactose,D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(−) fructose, D(−)mannitol, D(+) glucose, D(+) arabinose, D(−) arabinose, cellobiose, D(+)maltose, D(+) raffinose, L(>) rhamnose, D(+) melibiose, D(−) ribose,adonitol, D(+) arabitol, L(−) arabitol, D(+) fucose, L(−) fucose, D(−)lyxose, L(+) lyxose, and L(−) lyxose, or amino acids including, but notlimited to, glutamine, lysine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glycine, histidine, leucine, methionine,phenylalanine, proline, serine, threonine, tyrosine, valine, andtaurine. Methods and materials for enhancing blood brain barrierpenetration are described, e.g., in U.S. Pat. No. 4,866,042, “Method forthe delivery of genetic material across the blood brain barrier,” U.S.Pat. No. 6,294,520, “Material for passage through the blood-brainbarrier,” and U.S. Pat. No. 6,936,589, “Parenteral delivery systems,”all incorporated herein by reference in their entirety.

The subject antisense compounds may be admixed, encapsulated, conjugatedor otherwise associated with other molecules, molecule structures ormixtures of compounds, for example, liposomes, receptor-targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption. For example, cationic lipids maybe included in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is LIPOFECTIN (availablefrom GIBCO-BRL, Bethesda, Md.).

Oligonucleotides with at least one 2′-O-methoxyethyl modification arebelieved to be particularly useful for oral administration.Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carriers) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogeneous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 .mu m in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes that are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “stoically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids. When incorporated into liposomes, these specialized lipidsresult in liposomes with enhanced circulation lifetimes relative toliposomes lacking such specialized lipids. Examples of stericallystabilized liposomes are those in which part of the vesicle-forminglipid portion of the liposome comprises one or more glycolipids or isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. Liposomes and their uses are furtherdescribed in U.S. Pat. No. 6,287,860.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating nonsurfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein by reference.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein by reference. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bischloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drags and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. For example, the first targetmay be a particular antisense sequence of a Lipid transport andmetabolism gene, and the second target may be a region from anothernucleotide sequence. Alternatively, compositions of the invention maycontain two or more antisense compounds targeted to different regions ofthe same Lipid transport and metabolism gene nucleic acid target.Numerous examples of antisense compounds are illustrated herein andothers may be selected from among suitable compounds known in the art.Two or mote combined compounds may be used together or sequentially.

Dosing:

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drag accumulation in the body ofthe patient Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC50s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01.mu.g to 100 g per kg of body weight and may be given once or moredaily, weekly, monthly or yearly, or even once every 2 to 20 years.Persons of ordinary skill in the art can easily estimate repetitionrates for dosing based on measured residence times and concentrations ofthe drag in bodily fluids or tissues. Following successful treatment, itmay be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state, wherein the oligonucleotideis administered in maintenance doses, ranging from 0.01 .mu.g to 100 gper kg of body weight, once or more daily, to once every 20 years.

In embodiments, a patient is treated with a dosage of drag that is atleast about 1, at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, at least about 10, at least about 15, at least about 20,at least about 25, at least about 30, at least about 35, at least about40, at least about 45, at least about 50, at least about 60, at leastabout 70, at least about 80, at least about 90, or at least about 100mg/kg body weight. Certain injected dosages of antisenseoligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884,“Antisense modulation of PTP1B expression,” incorporated herein byreference in its entirety.

While various embodiments of (be present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document. Applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selectedembodiments of the invention. It will be appreciated that variations inproportions and alternatives in elements of the components shown will beapparent to those skilled in the art and are within the scope ofembodiments of the present invention.

Example 1 Design of Antisense Oligonucleotides Specific for a NucleicAcid Molecule Antisense to a Lipid Transport and Metabolism Gene and/ora Sense Strand of a Lipid Transport and Metabolism Gene Polynucleotide

As indicated above the term “oligonucleotide specific for” or“oligonucleotide targets” refers to an oligonucleotide having a sequence(i) capable of forming a stable complex with a portion of the targetedgene, or (ii) capable of forming a stable duplex with a portion of anmRNA transcript of the targeted gene.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced,Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the ease of in vitro assays.

The hybridization properties of the oligonucleotides described hereincan be determined by one or more in vitro assays as known in the art.For example, the properties of the oligonucleotides described herein canbe obtained by determination of binding strength between the targetnatural antisense and a potential drug molecules using melting curveassay.

The binding strength between the target natural antisense and apotential drug molecule (Molecule) can be estimated using any of theestablished methods of measuring the strength of intermolecularinteractions, for example, a melting curve assay.

Melting curve assay determines the temperature at which a rapidtransition from double-stranded to single-stranded conformation occursfor the natural antisense/Molecule complex. This temperature is widelyaccepted as a reliable measure of the interaction strength between thetwo molecules.

A melting curve assay can be performed using a cDNA copy of the actualnatural antisense RNA molecule or a synthetic DNA or RNA nucleotidecorresponding to the binding site of the Molecule. Multiple kitscontaining all necessary reagents to perform this assay are available(e.g. Applied Biosystems Inc. MeltDoctor kit). These kits include asuitable buffer solution containing one of the double strand DNA (dsDNA)binding dyes (such as ABI HRM dyes, SYBR Green, SYTO, etc.). Theproperties of the dsDNA dyes are such that they emit almost nofluorescence in free form, but are highly fluorescent when bound todsDNA.

To perform the assay the cDNA or a corresponding oligonucleotide aremixed with Molecule in concentrations defined by the particularmanufacturer's protocols. The mixture is heated to 95.degree. C. todissociate all pre-formed dsDNA complexes, then slowly cooled to roomtemperature or other lower temperature defined by the kit manufacturerto allow the DNA molecules to anneal. The newly formed complexes arethen slowly heated to 95.degree. C. with simultaneous continuouscollection of data on the amount of fluorescence that is produced by thereaction. The fluorescence intensity is inversely proportional to theamounts of dsDNA present in the reaction. The data can be collectedusing a real time PCR instrument compatible with the kit (e.g. ABI'sStepOne Plus Real Time PCR System or LightTyper instrument, RocheDiagnostics, Lewes, UK).

Melting peaks are constructed by plotting the negative derivative offluorescence with respect to temperature (-d(Fluorescence)/dT) on they-axis) against temperature (x-axis) using appropriate software (forexample LightTyper (Roche) or SDS Dissociation Curve, ABI). The data isanalyzed to identify the temperature of the rapid transition from dsDNAcomplex to single strand molecules. This temperature is called Tm and isdirectly proportional to the strength of interaction between the twomolecules. Typically, Tm will exceed 40.degree. C.

Example 2 Modulation of a Lipid Transport and Metabolism GenePolynucleotide Treatment of 518A2 Cells with Antisense Oligonucleotides

518A2 cells obtained from Albert Einstein-Montefiore Cancer Center, NYwere grown in growth media (MEM/EBSS (Hyclone cat # SH30024, orMediatech cat # MT-10-010-CV)+10% FBS (Mediatech cat #MT35-011-CV)+penicillin/streptomycin (Mediatech cat # MT30-002-CI)) at37.degree. C. and 5% CO2. One day before the experiment the cells werereplated at the density of 1.5.times. 105/ml into 6 well plates andincubated at 37.degree. C. and 5% CO2. On the day of the experiment themedia in the 6 well plates was changed to fresh growth media. Allantisense oligonucleotides were diluted to the concentration of 20 nM.Two .mu.l of this solution was incubated with 400 .mu.l of Opti-MEMmedia (Gibco cat #319854)70) and 4 .mu.l of Lipofectamine 2000(Invitrogen cat #11668019) at room temperature for 20 min and applied toeach well of the 6 well plates with 518A2 cells. A Similar mixtureincluding 2 .mu.l of water instead of the oligonucleotide solution wasused for the mock-transfected controls. After 3-18 h of incubation at37.degree. C. and 5% CO2 the media was changed to fresh growth media. 48h after addition of antisense oligonucleotides the media was removed andRNA was extracted from the cells using SV Total RNA Isolation Systemfrom Promega (cat #23105) or RNeasy Total RNA Isolation kit from Qiagen(cat #74181) following the manufacturers' instructions. 600 ng of RNAwas added to the reverse transcription reaction performed using VersocDNA kit from Thermo Scientific (cat # AB1453B) or High Capacity cDNAReverse Transcription Kit (cat #4368813 as described in themanufacturer's protocol. The cDNA from this reverse transcriptionreaction was used to monitor gene expression by real time PCR using ABITaqman Gene Expression Mix (cat #4369510) and primers/probes designed byABI (Applied Biosystems Taqman Gene Expression Assay by AppliedBiosystems Inc., Foster City Calif.). The following PCR cycle was used:50.degree. C. for 2 min, 95.degree. C. for 10 min, 40 cycles of(95.degree. C. for 15 seconds, 60.degree. C. for 1 min) using StepOnePlus Real Time PCR Machine (Applied Biosystems).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results

Real time PCR results show that the levels of ABCA1 mRNA in 518A2 cellsare significantly increased 48 h after treatment with one of the siRNAsdesigned to ABCA1 antisense AK311445 (FIG. 1A).

Real time PCR results show that the levels of ABCA1 mRNA in 518A2 cellsare significantly increased 48 h after treatment with six of the oligosdesigned to ABCA1 antisense AK311445 (FIG. 1B).

Treatment of 3T3 Cells with Antisense Oligonucleotides

3T3 cells from ATCC (cat # CRL-1658) were grown in growth media(MEM/EBSS (Hyclone cat # SH30024, or Mediatech cat # MT-10-010-CV)+10%FBS (Mediatech cat # MT35-011-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37.degree. C. and 5% CO.sub.2. One day before theexperiment the cells were replated at the density of 1.5.times.10.sup.5/ml into 6 well plates and incubated at 37.degree. C. and 5%CO.sub.2. On the day of the experiment the media in the 6 well plateswas changed to fresh growth media. All antisense oligonucleotides werediluted to the concentration of 20 .mu.M. Two .mu.l of this solution wasincubated with 400 .mu.l of Opti-MEM media (Gibco cat #31985-070) and 4.mu.l of Lipofectamine 2000 (Invitrogen cat #11668019) at roomtemperature for 20 min and applied to each well of the 6 well plateswith 3T3 cells. A Similar mixture including 2 .mu.l of water instead ofthe oligonucleotide solution was used for the mock-transfected controls.After 3-18 h of incubation at 37.degree. C. and 5% CO.sub.2 the mediawas changed to fresh growth media. 48 h after addition of antisenseoligonucleotides the media was removed and RNA was extracted from thecells using SV Total RNA Isolation System from Promega (cat # Z3105) orRNeasy Total RNA Isolation kit from Qiagen (cat #74181) following themanufacturers' instructions. 600 ng of RNA was added to the reversetranscription reaction performed using Verso cDNA kit from ThermoScientific (cal # AB1453B) or High Capacity cDNA Reverse TranscriptionKit (cat #4368813) as described in the manufacturer's protocol. The cDNAfrom this reverse transcription reaction was used to monitor geneexpression by real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by ABI (Applied Biosystems TaqmanGene Expression Assay by Applied Biosystems Inc., Foster City Calif.).The following PCR cycle was used: 50.degree. C. for 2 min, 95.degree. C.for 10 min, 40 cycles of (95.degree. C. for 15 seconds, 60.degree. C.for 1 min) using StepOne Plus Real Time PCR Machine (AppliedBiosystems).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results:

Real time PCR results show that the levels of ABCA1 mRNA in 3T3 cellsare significantly increased 48 h after treatment with three of theoligos designed to mouse ABCA1 antisense BF133827 (FIG. 1C).

Real Time PCR results show that levels of LRP1 mRNA in 3T3 cells aresignificantly increased 48 h after treatment with oligos to LRP1antisense DC401271 and AW544265 (FIG. 1J).

Treatment of HepG2 Cells with Antisense Oligonucleotides

Method 1: Treatment of HepG2 Cells with Naked AntisenseOligonucleotides:

HepG2 cells from ATCC (cat # HB-8065) woe grown in growth media(MEM/EBSS (Hyclone cat # SH30024, or Mediatech cal # MT-10-010-CV)+10%FBS (Mediatech cat # MT35-011-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37.degree. C. and 5% CO.sub.2. One day before theexperiment the cells were replated at the density of0.5.times.10.sup.4/ml into 6 well plates and incubated at 37.degree. C.and 5% CO.sub.2. On the day of the experiment the media in the 6 wellplates was replaced with 1.5 ml/well of fresh growth media. Allantisense oligonucleotides were diluted in water to the concentration of20 .mu.l of this solution was mixed with 400 .mu.l of fresh growth mediaand applied to each well of the 6 well plates with HepG2 cells. Asimilar mixture including 2 .mu.l of water instead of theoligonucleotide solution was used for the mock-treated controls. After3-18 h of incubation at 37.degree. C. and 5% CO.sub.2 the media waschanged to fresh growth media. 72 h after addition of antisenseoligonucleotides the cells were redosed as described in above. 48-72 hafter second dosing the media was removed and RNA was extracted from thecells using SV Total RNA Isolation System from Promega (cat # Z3105) orRNeasy Total RNA Isolation kit from Qiagen (cat #74181) following themanufacturers' instructions. 600 ng of RNA was added to the reversetranscription reaction performed using Verso cDNA kit from ThermoScientific (cat # AB1453B) as described in the manufacturer's protocol.Hie cDNA from this reverse transcription reaction was used to monitorgene expression by real time PCR using ABI Taqman Gene Expression Mix(cat #4369510) and primers/probes designed by ABI (Applied BiosystemsInc., Foster City Calif.). The following PCR cycle was used: 50.degree.C. for 2 min, 95.degree. C. for 10 min, 40 cycles of (95.degree. C. for15 seconds, 60.degree. C. for 1 min) using Mx4000 thermal cycler(Stratagene). Fold change in gene expression after treatment withantisense oligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Method Two: Treatment of HepG2 Cells with Antisense Oligonucleotides:

HepG2 cells from ATCC (cat # HB-8065) were grown in growth media(MEM/EBSS (Hyclone cat # SH30024, or Mediatech cal # MT-10-010-CV)+10%FBS (Mediatech cat # MT35-011-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37.degree. C. and 5% CO.sub.2. One day before theexperiment the cells were replated at the density of1.5.times.10.sup.5/ml into 6 well plates and incubated at 37.degree. C.and 5% CO.sub.2. On the day of the experiment the media in the 6 wellplates was changed to fresh growth media. All antisense oligonucleotideswere diluted to the concentration of 20 .mu.M. 2 .mu.l of this solutionwas incubated with 400 .mu.l of Opti-MEM media (Gibco cat #31985-070)and 4 .mu.l of Lipofectamine 2000 (Invitrogen cat #11668019) at roomtemperature for 20 min and applied to each well of the 6 well plateswith HepG2 cells. A similar mixture including 2 .mu.l of water insteadof the oligonucleotide solution was used for the mock-transfectedcontrols. After 3-18 h of incubation at 37.degree. C. and 5% CO.sub.2the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega (cat #Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat #74181)following the manufacturers' instructions. 600 ng of RNA was added tothe reverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cat # AB1453B) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat #4369510) and primers/probes designed by ABI(Applied Biosystems Inc., Foster City Calif.). The following PCR cyclewas used: 50.degree. C. for 2 min, 95.degree. C. for 10 min, 40 cyclesof (95.degree. C. for 15 seconds, 60.degree. C. for 1 min) using Mx4000thermal cycler (Stratagene). Fold change in gene expression aftertreatment with antisense oligonucleotides was calculated based on thedifference in 18S-normalized dCt values between treated andmock-transfected samples.

Results

Real time PCR results show that the levels of the LCAT mRNA in HepG2cells are significantly increased 48 h after treatment with two of theoligos designed to LCAT antisense Hs.668679 (FIG. 1E).

Real time PCR results show that the levels of the LCAT mRNA in HepG2cells are significantly increased 48 h after treatment with one of theoligos designed to LCAT antisense Hs.668679 (FIG. 1F).

Real Time PCR results show that levels of LRP1 mRNA in HepG2 cells aresignificantly increased 48 h after treatment with oligos to LRP1antisense DC401271 (FIG. 1H).

Real Time PCR results show that levels of LDLr mRNA in HepG2 cells aresignificantly increased 48 h after treatment with antisense oligos toLDLR antisense sherflor.aApr07.Oligos designed to LDLr antisensebloflor.aApr07 (CUR-1059-CUR-1063) did not elevate LDLr levels (FIGS. 1Kand 1L).Real time PCR results show that the levels of APOE mRNA in HepG2 cellsare significantly increased 48 h after treatment with three of theantisense oligos designed to APOE antisense Hs.626623. Oligos designedto APOE4 antisense Hs.714236 did not significantly elevate APOE mRNA(FIG. 1M).Real time PCR results show that the levels of ApoA1 mRNA in HepG2 cellsare significantly increased 48 h after treatment with some of theantisense oligonucleotides to ApoA1 antisense DA327409ext (FIG. 1N toFIG. 1P).Real time PCR results showing the fold change in ApoA1 mRNA (top panel)and ApoA1 natural antisense DA327409ext RNA (bottom panel) aftertreatment of HepG2 cells with naked LNA or phosphothioateoligonucleotides over 7 days as compared to control (FIG. 1Q).Real time PCR results showing the fold change in ApoA1 mRNA (orangebars) and ApoA1 natural antisense DA327409ext RNA (blue bars) aftertreatment of HepG2 cells with LNA oligonucleotides (FIG. 1R). Treatmentof Hek293 Cells with Antisense Oligonucleotides.

Hek293 cells from ATCC (cat # CRL-1573) were grown in growth media(MEM/EBSS (Hyclone cat # SH30024, or Mediatech cat # MT-10-010-CV)+10%FBS (Mediatech cat # MT35-011-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37.degree. C. and 5% CO.sub.2. One day before theexperiment the cells were replated at the density of1.5.times.10.sup.5/ml into 6 well plates and incubated at 37.degree. C.and 5% CO.sub.2. On the day of the experiment the media in the 6 wellplates was changed to fresh growth media. All antisense oligonucleotideswere diluted to the concentration of 20 .mu.M. Two .mu.l of thissolution was incubated with 400 .mu.l of Opti-MEM media (Gibco cat#31985-070) and 4 .mu.l of Lipofectamine 2000 (Invitrogen cat #11668019)at room temperature for 20 min and applied to each well of the 6 wellplates with Hek293 cells. A Similar mixture including 2 .mu.l of waterinstead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37.degree. C.and 5% CO.sub.2 the media was changed to fresh growth media. 48 h afteraddition of antisense oligonucleotides the media was removed and RNA wasextracted from the cells using SV Total RNA Isolation System fromPromega (cat # Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181) following the manufacturers' instructions. 600 ng of RNA wasadded to the reverse transcription reaction performed using Verso cDNAkit from Thermo Scientific (cat # AB1453B) or High Capacity cDNA ReverseTranscription Kit (cat #4368813) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat #4369510) and primers/probes designed by ABI(Applied Biosystems Taqman Gene Expression Assay by Applied BiosystemsInc., Foster City Calif.). The following PCR cycle was used: 50.degree.C. for 2 min, 95.degree. C. for 10 min, 40 cycles of (95.degree. C. for15 seconds, 60.degree. C. for 1 min) using using Mx4000 thermal cycler(Stratagene) or StepOne Plus Real Time PCR Machine (Applied Biosystems).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results:

Real Time PCR Results Show that the Levels of the LCAT mRNA in Hek293Cells are Significantly Increased 48 h after Treatment with Three of theOligos Designed to LCAT Antisense Hs.668679 (FIG. 1D).

Treatment of Vero 76 Cells with Antisense Oligonucleotides

Vero 76 cells from ATCC (cat # CRL-1587) were grown in growth media(MEM/EBSS (Hyclone cal # SH30024, or Mediatech cat # MT-10-010-CV)+10%FBS (Mediatech cat # MT35-011-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37.degree. C. and 5% CO.sub.2. One day before theexperiment the cells were replated at the density of1.3.times.10.sup.5/ml into 6 well plates and incubated at 37.degree. C.and 5% CO.sub.2. On the day of the experiment the media in the 6 wellplates was changed to fresh growth media. All antisense oligonucleotideswere diluted to the concentration of 20 .mu.M. Two .mu.l of thissolution was incubated with 400 .mu.l of Opti-MEM media (Gibco cat#31985-070) and 4 .mu.l of Lipofectamine 2000 (Invitrogen cat #11668019)at room temperature for 20 min and applied to each well of the 6 wellplates with Vero 76 cells. A Similar mixture including 2 .mu.l of waterinstead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37.degree. C.and 5% CO.sub.2 the media was changed to fresh growth media. 48 h afteraddition of antisense oligonucleotides the media was removed and RNA wasextracted from the cells using SV Total RNA Isolation System fromPromega (cat # Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181) following the manufacturers' instructions. 600 ng of RNA wasadded to the reverse transcription reaction performed using Verso cDNAkit from Thermo Scientific (cat # AB1453B) or High Capacity cDNA ReverseTranscription Kit (cat #4368813) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat #4369510) and primers/probes designed by ABI(Applied Biosystems Taqman Gene Expression Assay by Applied BiosystemsInc., Foster City Calif.). The following PCR cycle was used: 50.degree.C. for 2 min, 95.degree. C. for 10 min, 40 cycles of (95.degree. C. for15 seconds, 60.degree. C. for 1 min) using using Mx4000 thermal cycler(Stratagene) or StepOne Plus Real Time PCR Machine (Applied Biosystems).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results:

Real time PCR results show that the levels of the LCAT mRNA in Vetocells are significantly increased 48 h after treatment with one of theoligos designed to LCAT antisense Hs.668679 (FIG. 1G).

Real Time PCR results show that levels of LRP1 mRNA in Vero cells aresignificantly increased 48 h after treatment with oligos to LRP1antisense DC401271 and Hs.711951 (FIG. 11).

Detection Probes Used in Applied Biosystems Gene Expression Assays:

ABCA1: Hs00194045_ml (human), Mm01350760_ml (mouse)

LCAT: Hs00173415_ml

LRP1: Hs00233856_ml (human), Mm00464608_ml (mouse)

LDLR: Hs00181192_ml

ApoE: Hs00171168_ml

ApoA1: Hs00163641_ml. 18S cat #4319413E

Custom designed assay for ApoA1 antisense DA327409ext:

FAM Jabeled: TTTGGATCTGGACGACTTC (SEQ ID NO: 275)

Example 3 Modulation of a Lipid Transport and Metabolism Gene Expression

Materials and Methods

Cells were treated with either of the following methods:

Method 1: Treatment of HepG2 Cells with Naked AntisenseOligonucleotides:

HepG2 cell were grown in MEM/EBSS (Hyclone cal # SH30024)+10%FBS+penicillin+streptomycin at 37.degree. C. and 5% CO.sub.2. One daybefore the experiment the cells were replated at the density of1.5.times.10.sup.4/ml into 6 well plates and left at 37.degree. C. and5% CO.sub.2. On the day of the experiment the media in the 6 well plateswas changed to fresh MEM/EBSS+10% FBS. All antisense oligonucleotidesmanufactured by IDT were diluted to the concentration of 20 .mu.M. 2.mu.l of this solution was incubated with 400 .mu.l of Opti-MEM media(Gibco cat #31985-070) and applied to each well of the 6 well plateswith HepG2 cells. Similar mixture including 2 .mu.l of water instead ofthe oligonucleotide solution was used for the mock-transfected controls.72 h after addition of antisense oligonucleotides the media was removedand the dosing procedure was repeated as described in above.

48-72 h after repeated dosing RNA was extracted from the cells using SVTotal RNA Isolation System from Promega (cat # Z3105) or RNeasy TotalRNA Isolation kit from Qiagen (cat #74181) following the manufacturers'instructions. 600 ng of RNA was added to the reverse transcriptionreaction performed using Verso cDNA kit from Thermo Scientific (cat #AB1453B) as described in the manufacturer's protocol. The cDNA from thisreverse transcription reaction was used to monitor gene expression byreal lime PCR using ABI Taqman Gene Expression Mix (cat #4369510) andprimers/probes designed by ABI (Applied Biosystems Inc., Foster CityCalif.). The following PCR cycle was used: 50.degree. C. for 2 min,95.degree. C. for 10 min, 40 cycles of (95.degree. C. for 15 seconds,60.degree. C. for 1 min) using Mx4000 thermal cycler (Stratagene).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Primers and probe for the custom designed Taqman assay for the ApoA1natural antisense DA327409ext. Capital letters indicate unmodifieddeoxyribonucleotides

Probe sequence (FAM labeled) (SEQ ID NO: 275) TTTGGATCTGGACGACTTCForward Primer Seq. (SEQ ID NO: 276) CTCCTCCTGCCACTTCTTCTGReverse Primer Seq. (SEQ ID NO: 277) CTGGTGGATGAAGAAGGTTTGCMethod Two: Treatment of HepG2 Cells with Antisense Oligonucleotides:

HepG2 cells from ATCC (cat # HB-8065) were grown in growth media(MEM/EBSS (Hyclone cat # SH30024, or Mediatech cat # MT-10-010-CV)+10%FBS (Mediatech cal # MT35-011-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37.degree. C. and 5% CO.sub.2. One day before theexperiment the cells were replated at the density of1.5.times.10.sup.5/ml into 6 well plates and incubated at 37.degree. C.and 5% CO.sub.2. On the day of the experiment the media in the 6 wellplates was changed to fresh growth media. All antisense oligonucleotideswere diluted to the concentration of 20 .mu.M. 2 .mu.l of this solutionwas incubated with 400 .mu.l of Opti-MEM media (Gibco cat #31985-070)and 4 .mu.l of Lipofectamine 2000 (Invitrogen cat #11668019) at roomtemperature for 20 min and applied to each well of the 6 well plateswith HepG2 cells. Similar mixture including 2 .mu.l of water instead ofthe oligonucleotide solution was used for the mock-transfected controls.After 3-18 h of incubation at 37.degree. C. and 5% CO.sub.2 the mediawas changed to fresh growth media. 48 h after addition of antisenseoligonucleotides the media was removed and RNA was extracted from thecells using SV Total RNA Isolation System from Promega (cal #23105) orRNeasy Total RNA Isolation kit from Qiagen (cat #74181) following themanufacturers' instructions.

600 ng of RNA was added to the reverse transcription reaction performedusing Verso cDNA kit from Thermo Scientific (cat # AB1453B) as describedin the manufacturer's protocol. The cDNA from this reverse transcriptionreaction was used to monitor gene expression by real time FCR using ABITaqman Gene Expression Mix (cat #4369510) and primers/probes designed byABI (Applied Biosystems Inc., Foster City Calif.). The following PCRcycle was used: 50.degree. C. for 2 min, 95.degree. C. for 10 min, 40cycles of (95.degree. C. for 15 seconds, 60.degree. C. for 1 min) usingMx4000 thermal cycler (Stratagene). Fold change in gene expression aftertreatment with antisense oligonucleotides was calculated based on thedifference in 18S-normalized dCt values between treated andmock-transfected samples.

Primers and probe for the custom designed Taqman assay for the ApoA1natural antisense DA327409ext. Capital letters indicate unmodifieddeoxyribonucleotides

Probe sequence (FAM labeled) (SEQ ID NO: 275) TTTGGATCTGGACGACTTCForward Primer Seq. (SEQ ID NO: 276) CTCCTCCTGCCACTTCTTCTGReverse Primer Seq. (SEQ ID NO: 277) CTGGTGGATGAAGAAGGTTTGCTreatment of Primary Monkey Hepatocytes

Primary monkey hepatocytes were introduced into culture by RxGen Inc.and plated in 6 well plates. They were treated with oligonucleotides asfollows. The media in the 6 well plates was changed to fresh growthmedia consisting of William's Medium E (Sigma cat # W4128) supplementedwith 5% FBS, 50 U/ml penicillin and 50 ug/ml streptomycin, 4 ug/mlinsulin, 1 uM dexamethasone, 10 ug/ml Fungin (InVivogen, San DiegoCalif.). All antisense oligonucleotides were diluted to theconcentration of 20 .mu.M. 2 .mu.l of this solution was incubated with400 .mu.l of Opti-MEM media (Gibco cat #31985-070) and 4 .mu.l ofLipofectamine 2000 (Invitrogen cat #11668019) at room temperature for 20min and applied to each well of the 6 well plates with cells. Similarmixture including 2 .mu.l of water instead of the oligonucleotidesolution was used for the mock-transfected controls. After 3-18 h ofincubation at 37.degree. C. and 5% CO.sub.2 the media was changed tofresh growth media. 48 b after addition of antisense oligonucleotidesthe media was removed and RNA was extracted from the cells using SVTotal RNA Isolation System from Promega (cal # Z3105) or RNeasy TotalRNA Isolation kit from Qiagen (cat #74181) following the manufacturers'instructions. 600 ng of RNA was added to the reverse transcriptionreaction performed using Verso cDNA kit from Thermo Scientific (cat #AB1453B) as described in the manufacturer's protocol. The cDNA from thisreverse transcription reaction was used to monitor gene expression byreal time PCR using ABI Taqman Gene Expression Mix (cat #4369510) andprimers/probes designed by ABI (Applied Biosystems Inc., Foster CityCalif.). The following PCR cycle was used: 50.degree. C. for 2 min,95.degree. C. for 10 min, 40 cycles of (95.degree. C. for 15 seconds,60.degree. C. for 1 min) using Mx4000 thermal cycler (Stratagene). Foldchange in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.ELISA was conducted using MabTech Inc. ApoA1 ELISA kit cat #3710-11-6according to manufacturer's instructions.

The results are shown in FIG. 1Q to FIG. 1T. FIG. 1Q shows that botholigonucleotides with the phosphothioate backbone, i.e. internucleotidelinkages and LNA oligonucleotides were effective in modulating thetarget gene expression as measured by ApoA1 mRNA (top panel) and ApoA1antisense DA327409ext RNA (bottom panel) amounts detected. FIG. 1R showsthe levels of ApoA1 mRNA (orange bars) and ApoA1 antisense DA327409extRNA (blue bars) in HepG2 cells treated with oligonucleotides designedagainst DA327409ext. FIG. 1S shows dose dependent upregulation of ApoA1mRNA (bottom panel) and protein (top panel) in HepG2 cultures treatedwith oligonucleotides designed against DA327409ext. Fig. T showsupregulation of ApoA1 mRNA in primary African green monkey hepatocytesafter treatment with oligonucleotides designed against DA327409ext.

Example 4: Efficacy and Duration of Action Study of CUR-962 in theAfrican Green Monkey

The objective of this study was to assess and compare the effect ofantisense knockdown of the discordant noncoding antisense sequences thatregulate a Lipid transport and metabolism gene following intravenousadministration in a nonhuman primate model. The antisenseoligonucleotide test articles designed to inhibit the APOA1 regulatorysequences were designated as CUR-962.

CUR-962: (SEQ ID NO: 278) +G* + C*T* A*G*T* C*T*G* + T* + T* + GCUR-963 (control): (SEQ ID NO: 279) +G* + T*C* T*G*A* T*G*G* + A* + G* +ARegulatory Test Guidelines

This study was designed in accordance with accepted toxicologicalprinciples and to comply with International Conference of Harmonization(ICH) Harmonized Tripartite Guidelines (Non-Clinical Safety Studies forthe Conduct of Human Clinical Trials for Pharmaceuticals ICH M3(m), 2000Nov. 9), and generally accepted procedures for the testing oftherapeutic agents.

Test and Control Articles

Test Article Identity and Preparation

The test article, CUR-962, is a chemically stabilized antisenseoligonucleotide. The vehicle for intravenous delivery isphosphate-buffered saline (PBS).

Vehicle Characterization

For the PBS vehicle, the composition, batch number, expiry date andstorage conditions (temperature and light/dark) was obtained from thesupplier.

Test Article Storage and Handling

The test substance and vehicle were stored according to the receivedstorage conditions supplied by the Sponsor and manufacturer,accordingly.

Analysis of the Test Article Formulations

Samples of the test article formulation will be cryopreserved foranalysis of the concentration, stability and homogeneity of the testsubstance formulations.

Test System Rationale

The primate is a suitable non rodent species, acceptable to regulatoryauthorities as an indicator of potential hazards, and for whichextensive background data are available. The African green monkeyspecifically is a highly clinically relevant model of multiple humanphysiologic and disease states.

The intravenous route of administration corresponds to a possible humantherapeutic route. The dose of the test articles was based on theresults of the dose finding studies of analogous compounds previouslyperformed in the African green monkey.

African green monkey were chosen as the primate of choice as the testsubstances' target sequence are conserved across species with 100%homology in primates. Additionally, the test substance is a syntheticoligonucleotide. Consequently, dosing in primates allows for a superiorassessment of the efficacy of these compounds that would be morereflective of the uptake likely to be seen in humans than in any otherspecies.

Animals

Species: Chlorocebus sabaeus, non-human primate

Breed: African green monkey indigenous to St Kitts.

Source: RxGen, Lower Bourryeau, St. Kitts, West Indies.

Expected Age: The test animals were adults.

Expected Body Weight: The monkeys weigh approximately 3-4 kg. The actualrange may vary but will be documented in the data.

Sex: The test animals were adult females.

Number of Animals: Ten animals were screened to ensure identification of8 animals appropriate for enrollment in the study.

Number on Study: Females: 8

Justification for Number on Study:

This study was designed to use the fewest number of animals possible,consistent with the primary objective of evaluating the therapeuticefficacy of the test article in the African green monkey and priorstudies of the systemic administration of this type of oligonucleotidein this species.

Animal Specification:

Ten adult African Green monkeys in the weight range of 3 to 4 kg, wereemployed in the study. The monkeys were drug-naive adult animalshumanely trapped from the feral population that inhabits the island.Trapped monkeys were treated with anthelminthics to eliminate anypossible intestinal parasite burden and were observed in quarantine fora minimum of 4 weeks prior to screening for study enrollment. The age oftrapped monkeys were estimated by size and dentation, with the exclusionof older animals from the study. Prior to study enrollment, a clinicalexam was performed on each monkey, including evaluation of locomotionand dexterity. Blood samples were taken and sent to Antech Diagnostics(Memphis, Tenn.) for comprehensive clinical chemistries and a completeblood count and lipid profiles (see sections 9.2 and 319567928 forspecifications). Monkeys with abnormal lab values, as determined bycomparison to the established normal range for monkeys in the St. Kittscolony, were excluded from the study. In order to identify 8 monkeysthat satisfy this criterion, 10 monkeys were screened, with thescreening of additional animals as needed. Before study initiation, theselected monkeys will be transferred to individual cages to acclimate toindividual housing for a one-week period. Only animals deemed suitablefor experimentation will be enrolled in the study. The actual (orestimated) age and weight ranges at the start of the study will bedetailed in the raw data and final report

Animal Health and Welfare

The highest standards of animal welfare were followed and adhered toguidelines stipulated by the St. Kitts Department of Agriculture and theU.S. Department of Health and Human Services. All studies will beconducted in accordance with these requirements and all applicable codesof practice for the care and housing of laboratory animals. Allapplicable standards for veterinary care, operation, and review ascontained in the NIH Guide for the Care and Use of Animals. The St Kiltsfacility maintains an animal research committee that reviews theprotocols and inspects the facilities as required by the Guide. TheFoundation has an approved assurance filed with the Office of LaboratoryAnimal Welfare, as required by the Guide, M4384-01 (Axion ResearchFoundation/St. Kitts Biomedical Foundation). There are no specialnonhuman primate veterinary care issues and biohazard issues raised bythe research specified in this study.

Housing and Environment

To allow detection of any treatment-related clinical signs, the animalswere housed individually prior to surgery and postoperatively untilsacrifice. The primate building in which the individual cages weresituated were illuminated entirely by ambient light, which at 17 degreesnorth latitude approximates a 12 hr. 12 hr light-dark cycle asrecommended in the U.S. D.H.H.S guidelines. The RxGcn primate buildingwas completely ventilated to the outside. Additional air movement wasassured by ceiling fans to maintain a constant target temperature of23-35.degree. C., as is typical of St. Kitts throughout the year.Twenty-four hour extremes of temperature and relative humidity (whichalso will not be controlled) were measured daily. During the study, thecages were cleaned at regular intervals.

Diet and Water

Each animal was offered approximately 90 grams per day of a standardmonkey chow diet (TekLad, Madison, Wis.). The specific nutritionalcomposition of the diet was recorded. The water was periodicallyanalyzed for microbiological purity. The criteria for acceptable levelsof contaminants in stock diet and water supply were within theanalytical specifications established by the diet manufacturer and theperiodic facility water evaluations, respectively. The water met allcriteria necessary for certification as acceptable for humanconsumption.

Experimental Design

Animal Identification and Randomization

Allocation was done by means of a stratified randomization procedurebased on bodyweight and plasma cholesterol profiles. Prior to and afterallocation to a group, each animal was identified by a tattoo on theabdomen. Tattoos are placed on all colony animals as a means ofidentification in the course of routine health inspections. A cage planwas drawn up to identify the individuals housed within, and individualmonkeys were further identified by a labeled tag attached to theirrespective cage.

Group Sizes, Doses and Identification Numbers

The animals were assigned to 2 treatment groups, comprised of 4 monkey'sin each group. Specific animal identification numbers were provided toeach monkey according to the facility numbering system. This systemuniquely identifies each monkey by a letter followed by a three-digitnumber, e.g. Y032.

Route and Frequency of Administration

Animals were dosed once daily on Days 1, 3, and 5 deliveredintravenously by manual infusion over .about. 10 min. The infusion ratewill be 24 mL/kg/h. The animals were sedated with ketamine and xylazineprior to and during the dosing procedure. A venous catheter (Terumo minivein infusion set, 20 gauge needle, or similar appropriate infusion set)was inserted into the saphenous vein. Dosing took place in each monkeybetween 8:00 and 10:00 a.m. shortly after the animals wake and prior tofeeding. A blood sample to assess plasma cholesterol and other lipidlevels as described in Blood Chemistry section below, was collected justprior to each infusion. Blood collection preceded feeding at bothsampling intervals to minimize dietary effects on cholesterolmeasurements.

Clinical Observations

All visible signs of reaction to treatment were recorded on each day ofdosing. In addition, the animals were examined at least once each weekfor physical attributes such as appearance and general condition.

Body Weights

Body weights were recorded at weekly intervals during the treatment andpost-treatment periods.

Food Consumption

Individual food consumption was not be quantified. Feeding patterns,however, were be monitored and a note made of any major changes.

Mortality and Morbidity

Mortality and morbidity will be recorded. Any decision regardingpremature sacrifice will be made after consultation with the StudyDirector and with the Sponsor's Monitoring Scientist, if possible.Animals that are found dead or killed prematurely will be subjected tonecropsy with collection of liver, kidney, heart and spleen lung (issuesfor histopathology. In the event of premature sacrifice a blood samplewill also be taken (if possible) and the parameters determined. Animalsthat are found dead after regular working hours will be refrigeratedovernight and necropsies performed at the start of the next working day.If the condition of an animal requires premature sacrifice, it will beeuthanized by intravenous overdose of sodium pentobarbital. All researchis governed by the Principles for Use of Animals. RxGcn is required bylaw to comply with the U.S. Department of Health and Human Servicesstandards for primate facility, which dictates the levels of severitythat the procedures within this study, specified as mild, must abide.

Clinical Laboratory Studies

Blood Samples

Three blood samples were obtained from all animals prior to treatment,to establish a plasma cholesterol baseline. Blood samples were collectedpost treatment and were taken via superficial venipuncture. The volumecollected at any one sampling time point was not to exceed 8 ml, whichrepresents approximately 4% total blood volume of an adult monkey.

Animals had blood drawn at two baseline time points and on study days 1,3, 5, 7, 9, 11, 13 and 15, with continued weekly collection thereafteruntil total plasma cholesterol normalizes in group 1 (APOA1), if aperturbation is appreciated. Eight milliliters of blood were collectedon days 1, 6 and 11 to allow for assessment of clinical chemistries,lipid profiles and coagulation profiles. On all other days only 5 mls ofblood were collected, sufficient for clinical chemistries and lipidprofiles.

Blood samples were split into three parts on days on which bothchemistry and hematology measures will be made. One sample was collectedinto plasma collection tubes containing 25 .mu.l of heparin and labeledwith the study number, dose level, day number, date, unique animalidentification number. Following separation 1 ml of plasma was removedto a sterile cryotube carrying the above details and storedappropriately until shipment, for blood chemistry analysis. One aliquotof the plasma (0.5 ml) was removed to a sterile cryotube labeled withthe details described above and stored appropriately until shipment forplasma cholesterol distribution and Lipid profile analysis. Anadditional 1 ml and 0.5 ml aliquot of plasma was flash frozen and storedin liquid nitrogen to serve as back-up samples for potential additionalanalyses.

Two additional whole blood sample aliquots (2.5 ml each) were treatedwith acid citrate dextrose (ACD) anticoagulant and labeled, and storedat 4.degree. C. until shipped for coagulation and CBC measures detailedbelow.

The samples were shipped to arrive within 24 h of sampling, or storedunder stable conditions for shipment at a time determined appropriate.

Repeat samples were taken only if the method of sampling or the methodof assay was thought to be outside normal quality limits Samples weretaken into labeled tubes.

Hematology

A complete blood count (CBC), Prothrombin Time, PTT, Fibrinogen andD-Dimer were measured on all samples collected on days 1, 6 and 11 (andon additional days if perturbations are detected at any of these timepoints). Blood counts were assessed cm 1 ml of whole blood collected invacutainers containing EDTA. Coagulation profile determinations wereperformed on approximately 2.0 mL blood collected in vacutainerscontaining acid citrate dextrose (ACD) anticoagulant.

Blood Chemistry

Glucose, Blood Urea Nitrogen, Creatinine, Total protein, Albumin, Totalbilirubin. Alkaline Phosphatase, Alanine aminotransferase (ALT),Aspartate aminotransferase (AST), Cholesterol, Calcium, Phosphorus,Sodium, Potassium, Chloride, A/G ratio, BUN/Creatine (calculated)Globulins (calculated), Lipase, Amylase, Triglycerides, CPK, Lactatedehydrogenase, Gamma glutamyl transferase (GGT), Magnesium, TotalCholesterol LDL, VLDL, HDL, ApoA1, ApoA2, ApoB, ApoE, ApoLp(a).Superchemistries and LDL and HDL measures were made on every plasmasample. ApoA1 measures were made on select samples after assessment ofthe LDL and HDL data.

Determinations were performed on approximately 1.0 mL plasma for thesuperchemistry and 0.5 ml plasma for the cholesterol distribution andLipid transport and metabolism gene measures. An additional aliquot ofplasma was collected and stored for possible future analyses.

Liver Biopsies

A percutaneous liver biopsy was performed on all monkeys at baseline andon days 7 and 17. A 14 gauge biopsy needle (INRAD) will be employed toobtain 2 core biopsies (.about. 1.0 cm in length) from both the rightand left lobe of the liver. Successful biopsy was confirmed by visualinspection of the biopsy sample on the biopsy needle prior tosubdividing as indicated below.

The samples were pooled and then split in the following manner. Half ofone biopsy (.about.0.5 cm) from the left lobe was immersed inparaformaldehyde for sectioning for histopathology and in situ analysis.The remaining half of each of the divided biopsies, as well the othertwo intact biopsies were immediately immersed in a labeled cryotubecontaining 2 mls of RNAlater (Qiagen) and incubated at 4.degree. C.overnight, following which the RNAlater was aspirated and the sampletube flash frozen in liquid nitrogen. Following transportation in liquidnitrogen total RNA was isolated employing the Trizol or TriReagentmethod, with an expected yield of .about.40 .mu.g per 1.0 cm 14 g corebiopsy (.about.80-100 .mu.g total for the pooled RNA derived from all 4pooled core biopsies from a single monkey, absent the component savedfor histopathology and in situ). 5 .mu.g of the RNA fraction were usedfor target-specific real-time qPCR (TaqMan miRNA assay, ABI). Theremaining RNA fraction was reserved for possible genome wide expressionanalysis.

The fixed tissue was processed for paraffin embedding. Sections werestained for H&E and histopathological findings reported under GrossHistological findings. All slides generated in this work carried a labelwith the study number, dose level, day number, date, unique animalidentification number.

Statistical Analysis

Statistics

Descriptive statistics on hematology, clinical chemistries and lipidprofiles were performed. Appropriate bioinformatic analyses wasconducted on expression data.

Sample Size

Sample size determinations were made cm the basis of prior experimentsadministering modified antisense oligonucleotides to African greenmonkeys and resulting clinical chemistry and lipid profile changes andassociated variability. The total number of subjects for efficacyevaluation were twenty enrolled animals, with four animals per treatmentgroup, and four additional screened animals.

Results:

The results are shown in the following figures. FIG. 1U: ApoA1 mRNA (toppanels) and protein (bottom panels) levels increased in monkey liverbiopsies after treatment with CUR-962, an oligonucleotide designed toApoA1 antisense DA327409ext, compared to the baseline levels, asdetermined by real time PCR and ELISA respectively (two left panels).ApoA1 mRNA and protein levels did not change after the same period oftime in the control group dosed with an oligonucleotide that showed noeffect on ApoA1 levels in vitro (CUR-963, two right panels).

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may be desired and advantageous for any given orparticular application.

The Abstract of the disclosure will allow the reader to quicklyascertain the nature of the technical disclosure. It is submitted withthe understanding that it will not be used to interpret or limit thescope or meaning of the following claims.

What is claimed is:
 1. A compound comprising a single strandedsynthetic, modified antisense RNA oligonucleotide of 19 to 21nucleotides in length or an optionally modified double stranded siRNAcomprising an oligonucleotide of 19 to 30 nucleotides in length whereinsaid modifications are selected from: at least one modified sugarmoiety; at least one modified internucleotide linkage; at least onemodified nucleotide, and combinations thereof; wherein said siRNAoligonucleotide and said single stranded antisense RNA oligonucleotideare each 100% complementary with and specifically hybridize to a targetregion of a natural antisense polynucleotide of a lipid transport andmetabolism gene that is an ABCA1 polynucleotide wherein said naturalantisense polynucleotide is SEQ ID NO: 8, and where the antisense RNAoligonucleotide and the siRNA oligonucleotide upregulate the functionand/or expression of the ABCA1 polynucleotide in vivo or in vitro ascompared to a normal control, and wherein the target region in SEQ IDNO: 8 consists of nucleotides 1-245 and 799-1169, and further whereinthe siRNA oligonucleotide and the single stranded oligonucleotidecomprise a nucleobase sequence selected from the group consisting of SEQID NOS: 27, 28, 29, 31, 32, 33, 35, 38, and 39 wherein each thymine basemay be replaced by a uracil base.
 2. The siRNA oligonucleotide or singlestranded antisense RNA oligonucleotide of claim 1 comprising at leastone modified internucleotide linkage selected from the group consistingof phosphorothioate, alkylphosphonate, phosphorodithioate,alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphatetriester, acetamidate, carboxymethyl ester, and combinations thereof. 3.The siRNA oligonucleotide or single stranded antisense RNAoligonucleotide of claim 1 comprising at least one phosphorothioateinternucleotide linkage.
 4. The siRNA oligonucleotide or single strandedantisense RNA oligonucleotide of claim 1 comprising a backbone ofphosphorothioate internucleotide linkages.
 5. The siRNA oligonucleotideor single stranded antisense RNA oligonucleotide of claim 1 comprisingat least one modified nucleotide selected from: a peptide nucleic acid,a locked nucleic acid (LNA), an analogue thereof, a derivative thereof,and a combination thereof.
 6. The siRNA oligonucleotide or singlestranded antisense RNA oligonucleotide of claim 1 comprising a pluralityof modified internucleotide linkages selected from: phosphorothioate,alkylphosphonate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,carboxymethyl ester, and a combination thereof.
 7. The siRNAoligonucleotide or single stranded antisense RNA oligonucleotide ofclaim 1 comprising a plurality of modifications selected from peptidenucleic acids, locked nucleic acids (LNAs), analogues thereof,derivatives thereof, and a combination thereof.
 8. The siRNAoligonucleotide or single stranded antisense RNA oligonucleotide ofclaim 1 comprising at least one modified sugar moiety selected from: a2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugarmoiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugar moiety, anda combination thereof.
 9. The siRNA oligonucleotide or single strandedantisense RNA oligonucleotide of claim 1, wherein the oligonucleotidecomprises a plurality of modifications, wherein said modificationscomprise modified sugar moieties selected from: a 2′-O-methoxyethylmodified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkylmodified sugar moiety, a bicyclic sugar moiety, and a combinationthereof.
 10. A single stranded antisense oligonucleotide comprising anon-naturally occurring modified nucleotide wherein the nucleobasesequence of the oligonucleotide consists of a nucleobase sequenceselected from the group consisting of SEQ ID NOS: 27, 28, 29, 31, 32,33, 35, 38, 39, wherein each thymine base may be replaced with a uracilbase.
 11. A double stranded RNA oligonucleotide of 19 to 30 nucleotidesin length comprising the nucleobase sequence of SEQ ID NO: 23 or
 24. 12.A pharmaceutical composition comprising a double-stranded or singlestranded antisense oligonucleotide of 19 to 30 nucleotides in length anda pharmaceutically acceptable excipient, wherein the double-stranded orsingle stranded oligonucleotide comprises a non-naturally occurringnucleotide modification and the nucleobase sequence of any one of SEQ IDNOS; 23, 24, 27, 28, 29, 31, 32, 33, 35, 38 and 39, wherein each thyminebase may be replaced by a uracil base.
 13. The composition of claim 12,wherein the nucleobase sequence of the single stranded antisenseoligonucleotide is selected from the nucleobase sequence of any one ofSEQ ID NOS: 27, 28, 29, 31, 32, 33, 35, 38 and 39, wherein each thyminebase may be replaced by a uracil base.
 14. The composition of claim 12,wherein the double-stranded or single stranded antisense oligonucleotidecomprises the nucleobase sequence of SEQ ID NO: 23 or SEQ ID NO:
 24. 15.The composition of claim 12, wherein the double-stranded or singlestranded antisense oligonucleotide comprises one or more modificationsselected from: phosphorothioate, methylphosphonate, peptide nucleicacid, locked nucleic acid (LNA) molecules, and combinations thereof.